Anti-Phospholipase A2 Receptor Antibodies in the Diagnosis of Idiopathic Membranous Nephropathy

Circulating anti-phospholipase A2 receptor antibodies (anti-PLA2R) have been described in 70% to 80% of the patients with idiopathic membranous nephropathy (iMN), but not in patients with secondary membranous nephropathy or other glomerular diseases. The goal of this study was to evaluate the sensitivity and specificity of the assay for anti-PLA2R in the diagnosis of iMN. Anti-PLA2R IgG, Elisa and immunofluorescence tests were used to detect circulating anti-PLA2R. These tests were applied in 53 patients who had a kidney biopsy. Of these, 38 had histological diagnosis of membranous nephropathy (MN) and the remaining had other glomerular diseases. The MN was classified as idiopathic in 33 patients after clinical exclusion of secondary causes. Anti-PLA2R were positive in 57.6% of the patients with iMN. All patients with secondary membranous nephropathy or other glomerular diseases did not show circulating anti-PLA2R. The sensitivity was 57.6% (CI 39.2-74.5) and specificity 100% (CI 47.8-100), AUC 0.788; p < 0.0001 for the detection of iMN. 71.4% of the iMN patients that tested negative for anti-PLA2R were in partial or complete remission. The detection of anti-PLA2R in the studied population had a specificity of 100% for the iMN diagnosis. Prior treatments seem to make the test negative and contribute to a lower sensitivity.

Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs)

Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets. We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -‘good’ outcome no relapse > 3 years; ‘poor’ outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in ‘good’ outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression. We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments.

Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

Possible involvement of A1 receptors in the inhibition of gonadotropin secretion induced by adenosine in rat hemipituitaries in vitro

We investigated the participation of A1 or A2 receptors in the gonadotrope and their role in the regulation of LH and FSH secretion in adult rat hemipituitary preparations, using adenosine analogues. A dose-dependent inhibition of LH and FSH secretion was observed after the administration of graded doses of the R-isomer of phenylisopropyladenosine (R-PIA; 1 nM, 10 nM, 100 nM, 1 µM and 10 µM). The effect of R-PIA (10 nM) was blocked by the addition of 8-cyclopentyltheophylline (CPT), a selective A1 adenosine receptor antagonist, at the dose of 1 µM. The addition of an A2 receptor-specific agonist, 5-N-methylcarboxamidoadenosine (MECA), at the doses of 1 nM to 1 µM had no significant effect on LH or FSH secretion, suggesting the absence of this receptor subtype in the gonadotrope. However, a sharp inhibition of the basal secretion of these gonadotropins was observed after the administration of 10 µM MECA. This effect mimicked the inhibition induced by R-PIA, supporting the hypothesis of the presence of A1 receptors in the gonadotrope. R-PIA (1 nM to 1 µM) also inhibited the secretion of LH and FSH induced by phospholipase C (0.5 IU/ml) in a dose-dependent manner. These results suggest the presence of A1 receptors and the absence of A2 receptors in the gonadotrope. It is possible that the inhibition of LH and FSH secretion resulting from the activation of A1 receptors may have occurred independently of the increase in membrane phosphoinositide synthesis.

Stimulatory effects of adenosine on prolactin secretion in the pituitary gland of the rat

We investigated the effects of adenosine on prolactin (PRL) secretion from rat anterior pituitaries incubated in vitro. The administration of 5-N-methylcarboxamidoadenosine (MECA), an analog agonist that preferentially activates A2 receptors, induced a dose-dependent (1 nM to 1 µM) increase in the levels of PRL released, an effect abolished by 1,3-dipropyl-7-methylxanthine, an antagonist of A2 adenosine receptors. In addition, the basal levels of PRL secretion were decreased by the blockade of cyclooxygenase or lipoxygenase pathways, with indomethacin and nordihydroguaiaretic acid (NDGA), respectively. The stimulatory effects of MECA on PRL secretion persisted even after the addition of indomethacin, but not of NDGA, to the medium. MECA was unable to stimulate PRL secretion in the presence of dopamine, the strongest inhibitor of PRL release that works by inducing a decrease in adenylyl cyclase activity. Furthermore, the addition of adenosine (10 nM) mimicked the effects of MECA on PRL secretion, an effect that persisted regardless of the presence of LiCl (5 mM). The basal secretion of PRL was significatively reduced by LiCl, and restored by the concomitant addition of both LiCl and myo-inositol. These results indicate that PRL secretion is under a multifactorial regulatory mechanism, with the participation of different enzymes, including adenylyl cyclase, inositol-1-phosphatase, cyclooxygenase, and lipoxygenase. However, the increase in PRL secretion observed in the lactotroph in response to A2 adenosine receptor activation probably was mediated by mechanisms involving regulation of adenylyl cyclase, independent of membrane phosphoinositide synthesis or cyclooxygenase activity and partially dependent on lipoxygenase arachidonic acid-derived substances.

[Glomerulonephritis and vasculitis as causes of arterial hypertension]

The various types of glomerulonephritis, including many forms of vasculitis, are responsible for about 15% of cases of end-stage renal disease (ESRD). Arterial hypertension represents a frequent finding in patients suffering from glomerulonephritis or vasculitis and hypertension also serves as an indicator for these severe types of diseases. In addition, there are symptoms and signs like hematuria, proteinuria and renal failure. Especially, rapidly progressive glomerulonephritis (RPGN) constitutes a medical emergency and must not be missed by treating physicians. This disease can either occur limited to the kidneys or in the context of a systemic inflammatory disorder, like a vasculitis. If left untreated, RPGN can lead to a necrotizing destruction of glomeruli causing irreversible kidney damage within several months or even weeks. With respect to the immunologically caused vasculitis, there are - depending upon the severity and type of organ involved - many clinical warning signs to be recognized, such as arterial hypertension, hemoptysis, arthalgias, muscle pain, palpable purpura, hematuria, proteinuria and renal failure. In addition, constitutional signs, such as fever and loss of body weight may occur concurrently. Investigations of glomerulonephritis or vasculitis must contain a careful and complete examination of family history and medications used by the respective patient. Thereafter, a thorough clinical examination must follow, including skin, joints and measurement of arterial blood pressure. In addition, a spectrum of laboratory analyses is required in blood, such as full blood screen, erythrocyte sedimentation rate, CRP, creatinine, urea and glucose, and in urine, including urinalysis looking for hematuria, red cell casts and proteinuria. Importantly, proteinuria needs to be quantified by the utilization of a random urine sample. Proteinuria > 3g/d is diagnostic for a glomerular damage. These basic tests are usually followed by more specialized analyses, such as a screening for infections, including search for HIV, hepatitis B or C and various bacteria, and for systemic inflammatory diseases, including tests for antibodies, such as ANA, anti-dsDNA, ANCA, anti-GBM and anti-CCP. In cases of membranous nephropathy, antibodies against phospholipase-A2-receptor need to be looked for. Depending upon the given clinical circumstances and the type of disease, a reasonable tumor screening must be performed, especially in cases of membranous and minimal-change nephropathy. Finally, radiological examinations will complete the initial work-up. In most cases, at least an ultrasound of the kidney is mandatory. Thereafter, in most cases a renal biopsy is required to establish a firm diagnosis to define all treatment options and their chance of success. The elimination of a specific cause for a given glomerulonephritis or vasculitis, such as an infection, a malignancy or a drug-related side-effect, remains the key principle in the management of these diseases. ACE-inhibitors, angiotensin receptor-blockers, aldosteron antagonists and renin-inhibitors remain the mainstay in the therapy of arterial hypertension with proteinuria. Only in cases of persistently high proteinuria, ACE-inhibitors and angiotensin receptor blockers can be prescribed in combination. Certain types of glomerulonephritis and essentially all forms of vasculitis require some form of more specific anti-inflammatory therapy. Respective immunosuppressive drug regimens contain traditionally medications, such as glucocorticoids (e. g. prednisone), cyclosporine A, mycophenolate mofetil, cyclophosphamide, and azathioprine. With respect to more severe forms of glomerulonephritis and vasculitis, the antibody rituximab represents a new and less toxic alternative to cyclophosphamide. Finally, in certain special cases, like Goodpasture's syndrome or severe ANCA-positive vasculitis, a plasma exchange will be useful and even required.

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

Membranöse Glomerulopathie-eine Autoimmunerkrankung

Membranous nephropathy is one of the most common glomerular diseases and leading causes of nephrotic syndrome in Caucasian adults. Known as a clinico-pathologic entity for over 50 years, it is defined by thickening of the glomerular capillary membrane with subepithelial immuncomplexes. Secondary forms (e. g. hepatitis B, autoimmune disease or medication-induced) are distinguished from idiopathic forms. Despite spontaneous remissions in about 30 % of cases, one third of idiopathic forms progress to end-stage renal disease after 10 years. Seminal research progress of the last decade has allowed the identification of autoantibodies directed against podocytary elements leading to secondary damage to the filtration barrier. The so-called idiopathic membranous nephropathy has thus become a prototype of autoimmune disease. The autoantibodies detectable in 70 - 80 % of cases of idiopathic membranous nephropathy are directed against the M-type phospholipase A2-receptor on the podocyte membrane and correlate with disease activity. These epochal findings influence on diagnostic and therapeutic strategies establishing a rationale for the use of B cell-directed therapy on top of optimal supportive therapy.

Adenosine acts by A1 receptors to stimulate release of prolactin from anterior-pituitaries in vitro

Adenosine has been identified in the anterior pituitary gland and is secreted from cultured folliculostellate (FS) cells. To determine whether adenosine controls the secretion of anterior pituitary hormones in vitro, adenosine was incubated with anterior pituitaries. It stimulated prolactin (PRL) release at the lowest concentration used (10−10 M); the stimulation peaked at 10−8 M with a threefold increase in release and declined to minimal stimulation at 10−4 and 10−3 M. Follicle-stimulating hormone release was maximally inhibited at 10−8 M, whereas luteinizing hormone release was not significantly inhibited. Two selective A1 adenosine receptor antagonists (10−7 or 10−5 M) had no effect on basal PRL release, but either antagonist completely blocked the response to the most effective concentration of adenosine (10−8 M). In contrast, a highly specific A2 receptor antagonist (10−7 or 10−5 M) had no effect on basal PRL release or the stimulation of PRL release induced by adenosine (10−8 M). We conclude that adenosine acts to stimulate PRL release in vitro by activating A1 receptors. Since the A1 receptors decrease intracellular-free calcium, this would decrease the activation of nitric oxide synthase in the FS cells, resulting in decreased release of nitric oxide (NO). NO inhibits PRL release by activating guanylate cyclase that synthesizes cGMP from GTP; cGMP concentrations increase in the lactotrophs leading to inhibition of PRL release. In the case of adenosine, NO release from the FS cells decreases, resulting in decreased concentrations of NO in the lactotrophs, consequent decreased cGMP formation, and resultant increased PRL release.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) PKA-mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

Nicotinic acetylcholine receptors (nAChRs) are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

Decreased a2-adrenergic receptor in the brain stem and pancreatic islets during pancreatic regeneration in weanling rats

Sympathetic stimulation inhibits insulin secretion. a2-Adrenergic receptor is known to have a regulatory role in the sympathetic function. We investigated the changes in the a2-adrenergic receptors in the brain stein and pancreatic islets using [3H]Yohimbine during pancreatic regeneration in weanling rats. Brain stem and pancreatic islets of experimental rats showed a significant decrease (p<0.001) in norepinephrine (NE) content at 72 h after partial pancreatectomy. The epinephrine (EPI) content showed a significant decrease (p<0.001) in pancreatic islets while it was not detected in brain stem at 72 h after partial pancreatectomy. Scatchard analysis of [3H]Yohimbine showed a significant decrease (p<0.05) and Kd at 72 h after partial pancreatectomy in the brain stem. In the pancreatic islets, Scatchard analysis of [3H]Yohimbine showed a signiinfiBca'nnatx decrease (p<0.001) in B,nax and Kd (p<0.05) at 72 h after partial pancreatectomy. The binding parameters reversed to near sham by 7 days after pancreatectomy both in brain stein and pancreatic islets. This shows that pancreatic insulin secretion is influenced by central nervous system inputs from the brain stem. In vitro studies with yohimbine showed that the a2-adrenergic receptors are inhibitory to islet DNA synthesis and insulin secretion. Thus our results suggest that decreased a2-adrenergic receptors during pancreatic regeneration functionally regulate insulin secretion and pancreatic 13-cell proliferation in weanling rats.

Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.

Harmful drinking in military veterans with post-traumatic stress disorder: Association with the D2 dopamine receptor A1 allele

Aims: The frequency of the Taq I A alleles (A1 and A2) of the D2 dopamine receptor (DRD2) gene was examined in Caucasian post-traumatic stress disorder (PTSD) patients and controls. Results: In 91 PTSD patients, the frequency of the A1 allele was higher (P = 6.12 x 10(-3)) than in the 51 controls. In the 38 PTSD harmful drinkers (greater than or equal to60 g alcohol/day), A1 allelic frequency was higher (P = 3.91 x 10(-2)) than in the 53 non-harmful drinkers (