The frequency of anti-beta(2)-glycoprotein I antibodies is low and these antibodies are associated with pulmonary hypertension in mixed connective tissue disease

The objective of this study is to evaluate the prevalence of antiphospholipid antibodies, mainly anti-beta(2)-glycoprotein I (anti-beta(2)-GPI), and their possible clinical and laboratory relevance in mixed connective tissue disease (MCTD). This study included 39 consecutive patients with MCTD (Kasukawa`s criteria) from January, 2005, to March, 2007, and compared them with 21 age- and sex-matched healthy controls. IgG and IgM anticardiolipin (aCL) and anti-beta(2)-GPI were measured by ELISA. Lupus anticoagulant (LA) was detected by functional coagulation tests. Medium to high titres of aCL and anti-beta(2)-GPI antibodies were found in sera from four (10.2%) MCTD patients. One of these patients was found to be positive for IgM aCL, IgM anti-beta(2)-GPI and LA antibodies simultaneously. Additionally, this patient had a previous history of foetal loss in the second trimester and new-onset pulmonary arterial hypertension (PAH). The other three patients had none of the manifestations of antiphospholipid syndrome (APS) or PAH. The mean value of IgG anti-beta(2)-GPI was higher among those MCTD patients with PAH than in the group without PAH (34.2 +/- 46.8 vs 12.3 +/- 9.1, P = 0.018). None of the controls were positive for antiphospholipid antibodies. High to moderate titres of anti-beta(2)-GPI as well as APS were rare in MCTD, and these antibodies may be correlated with the development of PAH in these patients. Lupus (2009) 18, 618-621.

Val247Leu polymorphism of beta(2) glycoprotein 1 gene may justify the genesis of anti beta(2)GP1 antibodies and Antiphospholipid Syndrome in Multibacillary Leprosy

BACKGROUND - Multibacillary (MB) leprosy may be manifested with antiphospholipid antibodies (aPL), among which anti-beta(2)GP1 (beta(2)-glycoprotein 1). High titers of aPL are associated with APS (Antiphospholipid Syndrome), characterized by thrombosis. The mutation Val247Leu in the domain V of beta(2)GP1 exposes hidden epitopes with consequent development of anti-beta(2)GP1 antibodies. OBJECTIVE: To evaluate the Val247Leu polymorphism of beta(2)GP1 gene and its correlation with anti-beta(2)GP1 antibodies in leprosy patients. METHODS: The Val247Leu polymorphism was performed by PCR-RFLP and anti-beta(2)GP1 antibodies were measured by ELISA. RESULTS: The genotypic Val/Val was more prevalent in the leprosy group, compared to controls. Regarding the 7 MB patients with APS, four presented heterozygosis and three, Val/Val homozygosis. Although higher titrations of anti-beta(2)GP1 IgM antibodies were seen in MB leprosy group with Val/Leu and Val/Val genotypes, there was no statistical difference when compared to Leu/Leu genotype. CONCLUSION: The prevalence of Val/Val homozygosis in leprosy group can partially justify the presence of anti-beta(2)GP1 IgM antibodies in MB leprosy. The description of heterozygosis and Val/Val homozygosis in 7 patients with MB leprosy and thrombosis corroborates the implication of anomalous phenotype expression of beta(2)GP1 and development of anti-beta(2)GP1 antibodies, with consequent thrombosis and APS.

Val/Leu(247) Polymorphism of beta 2-glycoprotein I in Brazilian Patients with Antiphospholipid Syndrome-A Genetic Risk Factor?

A genetic polymorphism of the beta 2-glycoprotein I (beta 2-GPI) is recognized by antiphospholipid antibodies (aPL) and may even play a role in the development of antiphospholipid syndrome (APS). The objectives of this study were to determine a Val/Leu SNP at position 247 of the beta 2-GPI gene in Brazilian patients with APS and to compare these data with clinical and laboratory manifestations. Polymorphism assignment was performed by PCR followed by Rsa I restriction endonuclease. The titration of anti-beta 2-GPI antibodies was detected by ELISA. The results showed significantly higher frequencies of the V-encoding allele and the homozygous VV genotype in patients with APS than in control subjects (OR = 1.781, P = 0.0068; and OR = 6.413, P < 0.0001, respectively). The frequency of this genotype was also significantly higher in patients with arterial and venous thrombosis than in the control group (52% and 44%, respectively, versus 13%). Anti-beta 2-GPI-positive patients had significantly higher frequencies of the VV genotype than the controls subjects (OR = 8.179, P < 0.0001). These results suggest that the V-encoding allele and the homozygous VV genotype at position 247 of the beta 2-GPI gene may play a role in the generation of anomalous beta 2-GPI, with consequent auto-antibody production, and in phenotype expression of arterial and venous thrombosis in APS patients.

Correlation between beta-2-glycoprotein I gene polymorphism and anti-beta-2 glycoprotein I antibodies in patients with multibacillary leprosy

Antiphospholipid antibodies, such as anti-beta 2-glycoprotein I (beta 2GPI), are present in multibacillary leprosy (MB) patients; however, MB patients do not usually present with antiphospholipid antibody syndrome (APS), which is characterized by thromboembolic phenomena (TEP). Rare cases of TEP occur in leprosy patients, but the physiopathology of this condition remains unclear. In this case-control study, we examined whether single-nucleotide polymorphisms (SNPs) of the beta 2GPI gene contributed to the risk of leprosy and APS co-morbidity. SNPs Ser88Asn, Leu247Val, Cys306Gly and Trp316Ser were identified in 113 Brazilian leprosy patients. Additionally, anti-beta 2GPI antibodies and plasma concentrations of beta 2GPI were quantified. The Ser88Asn, Cys306Gly and Trp316Ser SNPs were not risk factors for APS in leprosy. A higher frequency of Val/Val homozygosity was observed in leprosy patients compared to controls (36 vs. 5%; P < 0.001). Forty-two percent of MB and 17% of paucibacillary leprosy patients were positive for anti-beta 2GPI IgM (P = 0.014). There was no correlation between SNP Ser88Asn or Cys306Gly and anti-beta 2GPI antibody levels. In MB patients with positive anti-beta 2GPI IgM, the frequency of Val/Val homozygosity was higher than in controls (32 vs. 15%; P = 0.042). The frequency of the mutant allele Ser316 was higher in MB patients with positive rather than negative anti-beta 2GPI IgM levels (6 vs. 0%; P = 0.040) and was greater than in the control group (6 vs. 1%; P = 0.034). The studied polymorphisms did not influence the plasma concentrations of beta 2GPI. These results suggest that Leu247Val and Trp316Ser SNPs may represent genetic risk factors for anti-beta 2GPI antibody production in MB patients.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Abolition of (-)-CGP 12177-evoked cardiostimulation in double beta(1)/beta(2)-adrenoceptor knockout mice. Obligatory role of beta(1)-adrenoceptors for putative beta 4-adrenoceptor pharmacology

Some beta (1)- and beta (2)-adrenoceptor-blocking agents, such as (-)-CGP 12177, cause cardiostimulant effects at concentrations considerably higher than those that antagonise the effects of catecholamines. The cardiostimulant effects of these non-conventional partial agonists are relatively resistant to blockade by (-)-propranolol and have been proposed to be mediated through putative beta (4)-adrenoceptors or through atypical states of either beta (1)- or beta (2)-adrenoceptors. We investigated the effects of (-)-CGP 12177 on sinoatrial rate and left atrial contractile force as well as the ventricular binding of (-)-[H-3]CGP 12177 in tissues from wild-type, beta (2)-adrenoceptor knockout and beta (1)/beta (2)-adrenoceptor double knockout mice. The cardiostimulant effects of (-)-CGP 12177 were present in wildtype and beta (2)-adrenoceptor knockout mice but were absent in beta (1)/beta (2)-adrenoceptor double knockout mice. Thus, the presence of beta (1)-adrenoceptors is obligatory for the cardiostimulant effects of (-)-CGP 12177. It appears therefore that an atypical state of the beta (1)-adrenoceptor contributes to the mediation of the cardiostimulant effects induced by non-conventional partial agonists. Ventricular beta (1)- and beta (2)-adrenoceptors, labelled in wild-type with a K(D)similar to0.5 nmol/l (similar to 16 fmol/mg protein), were absent in beta (1)/beta (2)-adrenoceptor double knockout mice. However, a high density binding site (similar to 154-391 fmol/mg protein) that did not saturate completely (K(D)similar to 80-200 nM) was labelled by (-)-[H-3]CGP 12177 in the three groups of mice, being distinct from beta (1)- and beta (2)-adrenoceptors, as well as from the site mediating the agonist effects of(-)-CGP 12177.

Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Functional beta(1)- and beta(2)-adrenoceptors in the left and right atrium of pre-hypertensive rats

There is a small increase in the functional beta(2)-adrenoceptor response on the spontaneously hypertensive rat (SHR) left atrium in the early stages of hypertension. In the present study, the functional beta(1)- and beta(2)-adrenoceptors of the left and right atrium in SHR pre-hypertension and age-matched (5-week-old) Wistar Kyoto (WKY) rats were characterized. Contractility methods with isoprenaline, T-0509 (a selective beta(1)-adrenoceptor agonist) and procaterol (a selective beta(2)-adrenoceptor agonist) were used. At 5 weeks, the SHRs were pre-hypertensive. Isoprenaline was more potent on the left atrium of 5-week-old SHRs than WKY rats. Bisoprolol, a selective beta(1)-adrenoceptor antagonist, was more potent against isoprenaline and T-0509 on the SHR than WKY rat left atrium. ICI 118,551, a selective beta(2)-adrenoceptor antagonist, was more potent against procaterol and T-0509 on the SHR than WKY rat left atrium. The results with bisoprolol and ICI 118,551 suggest that there are more functional beta(1)- and beta(2)-adrenoceptors on the left atrium of 5-week-old SHRs than WKY rats. Isoprenaline, T-0509 and procaterol were equipotent on the right atrium of 5-week-old WKY rats and SHRs. Bisoprolol was more potent against isoprenaline, T-0509 and procaterol on the SHR than WKY rat right atrium. ICI 118,551 was more potent against T-0509, but not isoprenaline and procaterol, on the SHR than WKY rat left atrium. This suggests there are more functional beta(1)-adrenoceptors, and probably more functional beta(2)-adrenoceptors, on the right atrium of 5-week-old SHRs than WKY rats. These functional differences in beta(1)-and beta(2)-adrenoceptor-mediated responses of the left and right atria of pre-hypertensive SHRs cannot be caused by hypertension, and may be associated with the onset of hypertension.

Stringent V beta requirement for the development of NK1.1+ T cell receptor-alpha/beta+ cells in mouse liver.

The liver of C57BL/6 mice contains a major subset of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells expressing the polymorphic natural killer NK1.1 surface marker. Liver NK1.1+TCR-alpha/beta+ (NK1+ T) cells require interaction with beta2-microglobulin-associated, major histocompatibility complex I-like molecules on hematopoietic cells for their development and have a TCR repertoire that is highly skewed to Vbeta8.2, Vbeta7, and Vbeta2. We show here that congenic C57BL/6.Vbeta(a) mice, which lack Vbeta8- expressing T cells owing to a genomic deletion at the Vbeta locus, maintain normal levels of liver NK1+ T cells owing to a dramatic increase in the proportion of cells expressing Vbeta7 and Vbeta2 (but not other Vbetas). Moreover, in C57BL/6 congenic TCR-V Vbeta3 and -Vbeta8.1 transgenic mice (which in theory should not express other Vbeta, owing to allelic exclusion at the TCR-beta locus), endogenous TCR-Vbeta8.2, Vbeta7, and Vbeta2 (but not other Vbetas) are frequently expressed on liver NK1+T cells but absent on lymph node T cells. Finally, when endogenous V beta expression is prevented in TCR-Vbeta3 and Vbeta8.1 transgenic mice (by introduction of a null allele at the C beta locus), the development of liver NK1+T cells is totally abrogated. Collectively, our data indicate that liver NK1+T cells have a stringent requirement for expression of TCR-Vbeta8.2, Vbeta7, or Vbeta2 for their development.

Multi-receptor binding profile of clozapine and olanzapine: a structural study based on the new beta (2) adrenergic receptor template

Schizophrenia is a devastating mental disorder that has a largeimpact on the quality of life for those who are afflicted and isvery costly for families and society.[1] Although the etiology ofschizophrenia is still unknown and no cure has yet beenfound, it is treatable, and pharmacological therapy often producessatisfactory results. Among the various antipsychoticdrugs in use, clozapine is widely recognized as one ofthemost clinically effective agents, even if it elicits significant sideeffects such as metabolic disorders and agranulocytosis. Clozapineand the closely related compound olanzapine are goodexamples ofdrug s with a complex multi-receptor profile ;[2]they have affinities toward serotonin, dopamine, a adrenergic,muscarinic, and histamine receptors, among others.

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta(2)-adrenergic receptor.

1. We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human beta(2) adrenergic receptor (beta(2)AR). 2. A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type beta(2)AR. 3. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4. We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the beta-ethanolamine tail with other residues of the beta(2)-AR binding pocket.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Negative antagonists promote an inactive conformation of the beta 2-adrenergic receptor.

The beta 2-adrenergic receptor undergoes isomerization between an inactive conformation (R) and an active conformation (R*). The formation of the active conformation of the receptor molecule can be promoted by adrenergic agonists or by mutations in the third cytoplasmic domain that constitutively activate the receptor. Here we show that, of several beta-adrenergic receptor-blocking drugs tested, only two, ICI 118551 and betaxolol, inhibit the basal signaling activity of the beta 2-adrenergic receptor, thus acting as negative antagonists. We document the molecular properties of the more efficacious ICI 118551; (i) it shows higher affinity for the inactive form of the receptor and (ii) it inhibits the spontaneous formation of a beta-adrenergic receptor kinase substrate by the receptor. These properties are opposite those of adrenergic agonists, indicating that, in a fashion reciprocal to that of agonists, negative antagonists promote the formation of an inactive conformation of the receptor.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.