Autoantibody from women with preeclampsia induces soluble Fms-like tyrosine kinase-1 production via angiotensin type 1 receptor and calcineurin/nuclear factor of activated T-cells signaling

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Recent evidence indicates that maternal endothelial dysfunction in preeclampsia results from increased soluble Fms-like tyrosine kinase-1 (sFlt-1), a circulating antiangiogenic protein. Factors responsible for excessive production of sFlt-1 in preeclampsia have not been identified. We tested the hypothesis that angiotensin II type 1 (AT1) receptor activating autoantibodies, which occur in women with preeclampsia, contribute to increased production of sFlt-1. IgG from women with preeclampsia stimulates the synthesis and secretion of sFlt-1 via AT1 receptor activation in pregnant mice, human placental villous explants, and human trophoblast cells. Using FK506 or short-interfering RNA targeted to the calcineurin catalytic subunit mRNA, we determined that calcineurin/nuclear factor of activated T-cells signaling functions downstream of the AT1 receptor to induce sFlt-1 synthesis and secretion by AT1-receptor activating autoantibodies. AT1-receptor activating autoantibody–induced sFlt-1 secretion resulted in inhibition of endothelial cell migration and capillary tube formation in vitro. Overall, our studies demonstrate that an autoantibody from women with preeclampsia induces sFlt-1 production via angiotensin receptor activation and downstream calcineurin/nuclear factor of activated T-cells signaling. These autoantibodies represent potentially important targets for diagnosis and therapeutic intervention.

Mechanical and material properties of cortical and trabecular bone from cannabinoid receptor-1-null (Cnr1-/-) mice

Funding ABK was funded by a studentship from the University of Aberdeen, Institute of Medical Sciences, and the Overseas Research Students Awards Scheme Acknowledgments We are grateful to Dr J.S. Gregory for assistance with Image J and Mr K. Mackenzie for assistance with Micro-CT analysis.

Type 1 ryanodine receptor interactions

Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research.

Peroxisome Proliferator-Activated Receptor-γ Coactivator 1-α (PPARGC1A) Genetic Associations with Type 2 Diabetes in Three Ethnicities

Genetic heterogeneity, lifestyle factors, gene-gene or gene-environment interactions are the determinants of T2D which puts Hispanics and populations with African ancestry at higher risk of developing T2D. In this dissertation, the genetic associations of PPARGC1A polymorphisms with T2D and its related phenotypes (metabolic markers) in Haitian Americans (cases=110, controls=116), African Americans (cases=120, controls=124) and Cuban Americans (cases=160, controls=181) of South Florida were explored. Five single nucleotide polymorphisms of gene PPARGC1A were evaluated in each ethnicity for their disease association. In Haitian Americans, rs7656250 (OR= 0.22, pp=0.03) had significant protective association with T2D but had risk association in African Americans for rs7656250 (OR=1.02, p=0.96) and rs4235308 (OR=2.53, p=0.03). We found that in Haitian American females, both rs7656250 (OR=0.23, pp=0.03) had protective association with T2D. In African American females, rs7656250 (OR=1.14, p=0.78) had risk association whereas in males, it had significant protective effect (OR=0.37, p=0.04). However, the risk association exhibited by rs4235308 was stronger in African American females (OR=2.69, p=0.03) than males (OR=1.16, p=0.72). In Cuban Americans, only rs7656250 showed significant risk association with T2D (OR=6.87, p=0.02) which was stronger in females alone (OR=7.67, p=0.01). We also observed significant differences among correlations of PPARGC1A SNPs and T2D phenotypes. Positive correlation was observed for log Hs-CRP with rs3774907 (pp=0.03) in Cuban Americans respectively. Correlation of log A1C with rs7656250 (p=0.02) was positive in Cuban Americans while it was negative for rs3774907 in Haitian Americans (ppPPARGC1A correlations with T2D and its phenotypes among the three ethnicities studied (ii) the associations of PPARGC1A SNPs showed significant effect modification by sex. The findings suggest that variations in effects of PPARGC1A gene polymorphisms among three ethnicities and between sexes may have biomedical implications for the development of T2D as well as the phenotypes related to T2D.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

DC-SIGN (CD209) gene promoter polymorphisms in a Brazilian population and their association with human T-cell lymphotropic virus type 1 infection

This study evaluated four polymorphisms located in the DC-SIGN (CD209) gene promoter region (positions -336, -332 -201 and -139) in DNA samples from four Brazilian ethnic groups (Caucasians, Afro-Brazilian, Asians and Amerindians) to establish the population distribution of these single-nucleotide polymorphisms (SNPs) and correlated DC-SIGN polymorphisms and infection in samples from human T-cell lymphotropic virus type 1 (HTLV-1)-infected individuals. To identify CD209 SNPs, 452 bp of the CD209 promoter region were sequenced and the genotype and allelic frequencies were evaluated. This is the first study to show genetic polymorphism in the CD209 gene in distinct Brazilian ethnic groups with the distribution of allelic and genotypic frequency. The results showed that -336A and -139A SNPs were quite common in Asians and that the -201T allele was not observed in Caucasians, Asians or Amerindians. No significant differences were observed between individuals with HTLV-1 disease and asymptomatic patients. However, the -336A variant was more frequent in HTLV-1 -infected patients [HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 80%; healthy asymptomatic HTLV-1 carriers, 90 %] than in the control group (70 %) [P=0.0197, odds ratio (OR)=2.511, 95 % confidence interval (CI)=1.218-5.179). In addition, the -139A allele was found to be associated with protection against HTLV-1 infection (P=0.0037, OR=0.3758, 95% CI=0.1954-0.7229) when the HTLV-1 -infected patients as a whole were compared with the healthy-control group. These observations suggest that the -139A allele may be associated with HTLV-1 infection, although no significant association was observed among asymptomatic and HAM/TSP patients. In conclusion, the variation observed in SNPs -336 and -139 indicates that this lectin may be of crucial importance in the susceptibility/transmission of HTLV-1 infections.

Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases

Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1 alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CK beta 8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals. J. Leukoc. Biol. 87: 885-893; 2010.

Renal response to the angiotensin II receptor subtype 1 antagonist irbesartan versus enalapril in hypertensive patients.

OBJECTIVE: To compare the acute and sustained renal hemodynamic effects on hypertensive patients of 100 mg irbesartan and 20 mg enalapril each once daily. PATIENTS: Twenty patients (aged 35-70 years) with uncomplicated, mild-to-moderate essential hypertension and normal serum creatinine levels completed this study. STUDY DESIGN: After random allocation to treatment (n=10 per group), administration schedule (morning or evening) was determined by further random allocation, with crossover of schedules after 6 weeks' therapy. Treatment and administration assignments were double-blind. Twenty-four-hour ambulatory blood pressure was monitored before and after 6 and 12 weeks of therapy. Renal hemodynamics were determined on the first day of drug administration and 12 and 24 h after the last dose during chronic treatment. RESULTS: Administration of each antihypertensive agent induced a renal vasodilatation with no significant change in glomerular filtration rate. However, the time course appeared to differ: irbesartan had no significant acute effect 4 h after the first dose, but during chronic administration a renal vasodilatory response was found 12 and 24 h after the dose; enalapril was effective acutely and 12 h after administration, but no residual effect was found 24 h after the dose. Both antihypertensive agents lowered mean ambulatory blood pressure effectively, with no significant difference between treatments or between administration schedules (morning versus evening). CONCLUSIONS: Irbesartan and enalapril have comparable effects on blood pressure and renal hemodynamics in hypertensive patients with normal renal functioning. However, the time profiles of the renal effects appear to differ, which might be important for long-term renoprotective effects.

IL2RA/CD25 gene polymorphisms: uneven association with multiple sclerosis (MS) and type 1 diabetes (T1D).

BACKGROUND: IL-2 receptor (IL2R) alpha is the specific component of the high affinity IL2R system involved in the immune response and in the control of autoimmunity. METHODS AND RESULTS: Here we perform a replication and fine mapping of the IL2RA gene region analyzing 3 SNPs previously associated with multiple sclerosis (MS) and 5 SNPs associated with type 1 diabetes (T1D) in a collection of 798 MS patients and 927 matched Caucasian controls from the south of Spain. We observed association with MS in 6 of 8 SNPs. The rs1570538, at the 3'- UTR extreme of the gene, previously reported to have a weak association with MS, is replicated here (P = 0.032). The most associated T1D SNP (rs41295061) was not associated with MS in the present study. However, the rs35285258, belonging to another independent group of SNPs associated with T1D, showed the maximal association in this study but different risk allele. We replicated the association of only one (rs2104286) of the two IL2RA SNPs identified in the recently performed genome-wide association study of MS. CONCLUSIONS: These findings confirm and extend the association of this gene with MS and reveal a genetic heterogeneity of the associated polymorphisms and risk alleles between MS and T1D suggesting different immunopathological roles of IL2RA in these two diseases.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

CB1 cannabinoid receptor modulates MDMA acute responses and reinforcement

Background: 3, 4-methylenedioxymethamphetamine (MDMA) is a popular recreational drug widely abused by young people. The endocannabinoid system is involved in the addictive processes induced by different drugs of abuse. However, the role of this system in the pharmacological effects of MDMA has not been yet clarified.Methods: Locomotion, body temperature and anxiogenic-like responses were evaluated after acute MDMA administration in CB1 knockout mice. Additionally, MDMA rewarding properties were investigated in the place conditioning and the intravenous self-administration paradigms. Extracellular levels of DA in the nucleus accumbens were also analyzed after a single administration of MDMA by in vivo microdialysis. Results: Acute MDMA administration increased locomotor activity, body temperature and anxiogenic-like responses in wild type mice, but these responses were lower or abolished in knockout animals. MDMA produced similar conditioned place preference and increased dopamine extracellular levels in the nucleus accumbens in both genotypes. Nevertheless, CB1 knockout mice failed to self-administer MDMA at any of the doses used. Conclusions: These results indicate that CB1 cannabinoid receptors play an important role in the acute prototypical effects of MDMA, and are essential in the acquisition of an operant behavior to self-administer this drug.

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension.

OBJECTIVES: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. METHODS: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. RESULTS: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. CONCLUSIONS: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.