Is serum amyloid A an endogenous TLR4 agonist?

Serum amyloid A (SAA), a classical acute-phase protein, is produced predominantly by hepatocytes in response to injury, infection, and inflammation. It has been shown that SAA primes leukocytes and induces the expression and release of proinflammatory cytokines. Here, we report that SAA induces NO production by murine peritoneal macrophages. Using specific inhibitors, we showed that NO production was dependent on inducible NO synthase thorough the activation of ERK1/2 and p38 MAPKs. Moreover, SAA activity was decreased after proteolysis but not with polymyxin B, a lipid A antagonist. Finally, we found that NO production was dependent on functional TLR4, a receptor complex associated with innate immunity. Macrophages from C3H/HeJ and C57BL/10ScCr mice lacking a functional TLR4 did not respond to SAA stimulation. In conclusion, our study makes a novel observation that SAA might be an endogenous agonist for the TLR4 complex on macrophages. The contribution of this finding in amplifying innate immunity during the inflammatory process is discussed.

Paclitaxel associated with cholesterol-rich nanoemulsions promotes atherosclerosis regression in the rabbit

A cholesterol-rich nanoemulsion (LDE) that resembles LDL binds to the LDL receptors and after injection into the blood stream may concentrate in cells with LDL receptor overexpression, as occurs in neoplasias and other proliferative processes. Thus, LDE can be used as vehicle to target drugs against those cells. The current study was designed to verify in rabbits whether LDE concentrates in the lesioned rabbit artery and whether a paclitaxel derivative, paclitaxel oleate, associated to LDE could reduce the atherosclerotic lesions. Sixteen male New Zealand rabbits were fed a 1% cholesterol diet for 60 days. Starting from day 30 under cholesterol feeding, eight animals were treated with four weekly intravenous injections of LDE-paclitaxel (4 mg/kg) and eight with four weekly intravenous saline solution injections for additional 30 days. On day 60, the animals were sacrificed for analysis. The uptake of LDE labeled with [C-14]-cholesteryl oleate by the aortic arch of cholesterol-fed rabbits was twice as much that observed in animals fed only regular chow. LDE-paclitaxel reduced the lesion areas of cholesterol-fed animals by 60% and intima-media ratio fourfold and inhibited the macrophage migration and the smooth muscle cell proliferation and invasion of the intima. LDE-paclitaxel treatment had no toxicity. In conclusion, LDE-paclitaxel produced pronounced atherosclerosis regression without toxicity and has shown remarkable potential in cardiovascular therapeutics. (c) 2008 Published by Elsevier Ireland Ltd.

Increased MMA concentration and body mass index are associated with spontaneous abortion in Brazilian women A pilot study

Background: The pathophysiology of spontaneous abortion is complex and may involve the interaction of genetic and environmental factors. We evaluated the predictors of spontaneous abortion in Brazilian pregnant women. The effects of age, gestational age. body mass index (BMI), cigarette smoking, alcohol ingestion, use of multivitamins and concentrations of vitamins (folate, cobalamin and vitamin 136) and vitamin-dependent metabolites were analyzed. Methods: Study population included 100 healthy women that attended pre-natal care in 2 health centers of Sao Paulo, Brazil, and in whom pregnancy outcome was known. Folate and cobalamin status was measured in blood specimens collected between 4 and 16 weeks. The genotypes for 8 gene polymorphisms were evaluated by PCR-RFLP. Results: Eighty-eight women had normal pregnancy outcome (Group 1), while 12 experienced a miscarriage after blood collection (Group 2). Increased methylmalonic acid (MMA) concentrations were found in Group 2 (median [25th-75th percentile]=274 [149-425] nmol/l) relative to Group 1 (138 [98-185]) (P<0.01). No differences between the groups were observed for serum cobalamin, serum or red cell folate, and serum total homocysteine or allele frequencies for 8 polymorphisms. In a conditional logistic regression analysis including age, gestational age, serum creatinine, MMA, cystathionine, body mass index (BMI), cigarette smoking, alcohol ingestion and use of multivitamins the risk of abortion was significantly associated with MMA (OR [95% CI] = 3.80 [1.36, 10.62] per quartile increase in MMA), BMI (OR [95% CI] = 5.49 [1.29,23.39] per quartile) and gestational age (OR [95% CI] = 0.10 [0.01, 0.77] per increase of interval in gestational age). Conclusions: Increased serum MMA and BMI concentrations are associated with spontaneous abortion in Brazilian women. (C) 2009 Elsevier B.V. All rights reserved.

Human Airway Epithelial Cell Responses to Neisseria lactamica and Purified Porin via Toll-Like Receptor 2-Dependent Signaling

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin`s surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

Transcript profiling of papaya fruit reveals differentially expressed genes associated with fruit ripening

Papaya (Carica papaya L) fruit has a short shelf life due to fast ripening induced by ethylene, but little is known about the genetic control of ripening and attributes of fruit quality. Therefore, we identified ripening-related genes affected by ethylene using cDNA-AFLP (Amplified Fragment Length Polymorphism of cDNA). Transcript profiling of non-induced and ethylene-induced fruit samples was performed, and 71 differentially expressed genes were identified. Among those genes some involved in ethylene biosynthesis, regulation of transcription, and stress responses or plant defence were found (heat shock proteins, polygalacturonase-inhibiting protein, and acyl-CoA oxidases). Several transcription factors were isolated, and except for a 14-3-3 protein, an AP2 domain-containing factor, a salt-tolerant zinc finger protein, and a suppressor of PhyA-105 1, most of them were negatively affected by ethylene, including fragments of transcripts similar to VRN1, and ethylene responsive factors (ERF). With respect to fruit quality, genes related to cell wall structure or metabolism, volatiles or pigment precursors, and vitamin biosynthesis were also found. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Genetic analysis of complement C1s deficiency associated with systemic lupus erythernatosus highlights alternative splicing of normal C1s gene

Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of Clr has been observed to occur concomitantly with deficiency in Cls and 9 out of 15 reported cases presented systemic lupus erythernatosus (SLE). Here, we describe a family in which all four children are deficient in Cls but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children`s sera. Cls was undetectable, while in the parents` sera it was lower than in the normal controls. The levels of Clr observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of Cls synthesis was observed in the patients` fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the Cls cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of Cls mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3` splice site within intron I which increases the size of exon 2 by 87 nucleotides. (c) 2007 Elsevier Ltd. All rights reserved.

Increase of core temperature induced by corticotropin-releasing factor and urocortin: A comparative study

This study compared the ability of CRF and UCN1 to induce a thermoregulatory response when centrally injected into rats. The effects of antipyretic drugs and CRF receptor antagonists (CRF(1) and CRF(2)) on the temperature (T) changes induced by these peptides were also investigated. Rectal (rT) and tail skin (T(sk)) temperatures were measured with a thermistor probe while body (bT) temperature was measured with a battery-operated biotelemetry transmitter in male Wistar rats (200 g) every 30 min over a period of 6 h, after intracerebroventricular (i.c.v.) injection of 1 nmol of either CRF or UCN1. Rats were pre-treated with indomethacin (2 mg kg(-1), i.p.) or celecoxib (5 mg kg(-1), p.o.), dexamethasone (0.5 mg kg(-1), s.c.), astressin (a CRF(1)/CRF(2) antagonist, 7 nmol, icy.) or antalarmin (a CRF(1) antagonist, 20 mg kg 1, i.p.). The increase in body temperature induced by CRF was accompanied by a reduction in T(sk) while the response induced by UCN1 was accompanied by an elevation in T(sk). Indomethacin or celecoxib did not change the increases in rT caused by either CRF or UCN1. Although dexamethasone attenuated the increase in rectal temperature in response to CRF, dexamethasone did not modify the response induced by UCN1. Astressin blocked the UCN1-induced hyperthermia and reduced CRF-induced fever. Antalarmin did not modify the hyperthermia in response to UCN1, but reduced the fever evoked by CRF. This study demonstrated that CRF by acting on the CRF(1) receptor induces a prostaglandin-independent fever which seems to depend, at least in part, on the synthesis of other mediators while UCN1 acts on the CRF(2) receptor, promoting a hyperthermic response which seems to be independent on synthesis/release of any mediator. (C) 2010 Elsevier B.V. All rights reserved.

Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB(4) induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB(4) is also important in early effector T cell recruitment that is mediated by LTB(4) receptor 1, the high-affinity receptor for LTB(4). The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB(4) and IFN-gamma production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase(-/-), a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-beta as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase(-/-)-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis. The Journal of Immunology, 2008,181: 8544-8551.

Dexamethasone Effects in the Strongyloides venezuelensis Infection in A Murine Model

The aim of this study was to investigate the immunomodulatory effects of glucocorticoids on the immune response to Strongyloides venezuelensis in mice. Balb/c mice were infected with S. venezuelensis and treated with Dexamethasone (Dexa) or vehicle. Dexa treatment increased circulating blood neutrophil numbers and inhibited eosinophil and mononuclear cell accumulation in the blood, bronchoalveolar, and peritoneal fluid compared with control animals. Moreover, Dexa decreased tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-3 (IL-3), IL-4, IL-5, IL-10, and IL-12 production in the lungs and circulating immunoglobulin G1. (IgG1), IgG2a, and IgE antibody levels while increasing the overall parasite burden in the feces and intestine. Dexa treatment enhanced the fertility of female nematodes relative to untreated and infected mice. In summary, the alterations in the immune response induced by Dexa resulted in a blunted, aberrant immune response associated with increased parasite burden. This phenomenon is similar to that observed in S. stercoralis-infected humans who are taking immunosuppressive or antiinflammatory drugs, including corticosteroids.

16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects

Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the α-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggests that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized.

The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF 1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2, mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

Insulin-regulatable phosphoproteins in 3T3-L1 adipocytes form detergent-insoluble complexes not associated with caveolin

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, som: of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected P-32-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.

Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS

The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A valiant mRNA Tvas identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount irt situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice

Previously we described activating mutations of h beta(c), the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, h beta(c)FI Delta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the h beta(c)FI Delta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in h beta(c) may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic h beta(c) activation has the potential to contribute to pathological events in the central nervous system.