Multicentric evaluation of a specific intrathecal anti-Treponema pallidum IgG index as a diagnostic biomarker of neurosyphilis: results from a retro-prospective case-control study.

BACKGROUND AND OBJECTIVES: The diagnosis of neurosyphilis (NS) lacks a true 'gold standard', making the diagnosis challenging while consequences of a misdiagnosis are potentially severe. The aim of this study was to evaluate the diagnostic performance of measuring an antibody index (AI) for the intrathecal synthesis of specific anti-Treponema pallidum (T. pallidum) IgG for the diagnosis of NS. METHODS: Specific anti-T. pallidum IgG were measured simultaneously in paired cerebrospinal fluid (CSF)-serum samples collected retrospectively and prospectively between 2007 and 2022, from patients suspected of NS, in Switzerland. An AI was calculated to account for blood-brain barrier integrity. Area under the receiver operating characteristic curve, sensitivity/specificity and positive/negative predictive values of AI test were estimated. Two NS definitions were used: NS1 included patients with NS suspicion presenting with neurological symptoms and/or acute neurosensory signs, and positive T. Pallidum Hemagglutinations Assay (TPHA)/T. pallidum particle agglutination assay (TPPA) serology and CSF-TPHA/TPPA ≥320, and either CSF-leucocytes >5 cells/mm3 and/or CSF-protein >0.45 g/L and/or a reactive CSF-venereal disease research laboratory (VDRL)/rapid plasma reagin (RPR) test. NS2 included patients with suspected NS presenting with acute ocular and/or otologic symptoms, and positive TPHA/TPPA serology, and a favourable response to NS treatment. Controls were patients diagnosed with any other central nervous system (CNS) pathologies and with positive TPHA/TPPA serology. RESULTS: The study included 71 NS (43 NS1 and 28 NS2) and 110 controls. With a threshold of ≥1.7, sensitivity and specificity of the specific AI test were 90.7% (CI 77.7 to 97.4) and 100% (CI 96.7 to 100.0), respectively, for NS1 and 14.3% (CI 4 to 32.7) and 100% (CI 96.7 to 100.0) for NS2. In patients suspected of NS with a CNS involvement (NS1 group), NS could be confirmed by the positivity of this specific AI. CONCLUSIONS: Measurement of an intrathecal synthesis index of specific anti-T. pallidum IgG in patients with CSF inflammatory signs appears to be a valuable diagnostic test. However, in otic or ocular syphilis, presenting few CSF abnormalities, AI is not sufficient alone to confirm NS diagnosis. TRIAL REGISTRATION: Swiss Association of Research Ethics Committees number 2019-00232.

Cerebrospinal Fluid Features in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Reverse Transcription Polymerase Chain Reaction (RT-PCR) Positive Patients.

This study analyzed the cerebrospinal fluid features of 31 coronavirus disease 2019 (COVID-19) patients with neurological complications. We observed neither severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the cerebrospinal fluid, nor intrathecal immunoglobulin G (IgG) synthesis but did observe signs of blood-brain barrier disruption. These results might serve as a basis for a better understanding of SARS-CoV-2 related neuropathogenesis.

A novel series of metabotropic glutamate receptor 5 negative allosteric modulators based on a 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine core.

A series of potent non-acetylinic negative allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5 NAMs) was developed starting from HTS screening hit 1. Potency was improved via iterative SAR, and physicochemical properties were optimized to deliver orally bioavailable compounds acceptable for in vivo testing. A lead molecule from the series demonstrated dose-dependent activity in the second phase of the rat formalin test from 30 mg/kg, and a preliminary PK/PD relationship was established.

Intrathecal Synthesis Index of Specific Anti-Treponema IgG: a New Tool for the Diagnosis of Neurosyphilis.

Neurosyphilis (NS) diagnosis is challenging because clinical signs are diverse and unspecific, and a sensitive and specific laboratory test is lacking. We tested the performance of an antibody index (AI) for intrathecal synthesis of specific anti-Treponema IgG by enzyme-linked immunosorbent assay (ELISA) for NS diagnosis. We conducted a retroprospective monocentric study including adults with neurological symptoms who had serum and cerebral spinal fluid (CSF) samples collected between 2006 and 2021. Two NS definitions were used. NS1 included patients with neurological symptoms, positive Treponema pallidum particle agglutination (TPPA) serology, and CSF-TPPA of ≥320, as well as CSF-leukocytes of >5 cells/mm3 and/or CSF-protein of >0.45 g/L and/or a reactive CSF-VDRL/RPR test. NS2 included patients with acute ocular and/or otologic symptoms, positive TPPA serology, and a response to NS treatment. Controls were patients with central nervous system disorders other than neurosyphilis. Anti-Treponema pallidum IgG were measured simultaneously in serum and CSF, and AI was calculated according to Reiber diagram. We assessed the AI test area under the curve (AUC), sensitivity/specificity, and estimated positive and negative predictive values. In total, 16 NS1 patients, 11 NS2 patients, and 71 controls were included. With an AI of ≥1.7 as a positive test for NS diagnostic, specificity was 98.6% (95% confidence interval [CI 95%] of 92.4 to 100.0) and sensitivity was 81.3% (CI 95% of 54.4 to 96.0) for NS1 and 98.6% (CI 95% 92.4 to 100.0) and 27.3% (CI 95% 6.0 to 61.0), respectively, for NS2. Positive and negative predictive values were >95% for NS1 and >85% for NS2, for prevalence above and below 20%. Measuring an AI for intrathecal synthesis of specific anti-Treponema pallidum IgG is a new promising tool highly specific for NS diagnosis. IMPORTANCE In the context of a lack of a gold standard for the diagnosis of neurosyphilis due to either nonspecific or nonsensitive tests, we present in this article a new promising tool highly specific for NS diagnosis. This new test involves measuring an intrathecal synthesis index of specific anti-Treponema IgG by ELISA.

Characterization of an mGluR2/3 negative allosteric modulator in rodent models of depression.

There is growing evidence suggesting that antagonists of group II metabotropic glutamate receptors (mGluR2/3) exhibit antidepressant-like properties in several preclinical models of depression. However, all those studies have been performed using competitive group II non-selective orthosteric antagonists. In this study we extensively characterized a group II selective negative allosteric modulator (4-[3-(2,6-Dimethylpyridin-4-yl)phenyl]-7-methyl-8-trifluoromethyl-1,3-dihydrobenzo[b][1,4]diazepin-2-one, namely RO4491533, Woltering et al., 2010) in several in vitro biochemical assays and in vivo models of depression. In vitro, RO4491533 completely blocked the glutamate-induced Ca(2+) mobilization and the glutamate-induced accumulation in [(35)S]GTP(γS) binding in cells expressing recombinant human or rat mGluR2 and in native tissues. Results from Schild plot experiments and reversibility test at the target on both cellular and membrane-based assays confirmed the negative allosteric modulator properties of the compound. RO4491533 was equipotent on mGluR2 and mGluR3 receptors but not active on any other mGluRs. RO4491533 has acceptable PK properties in mice and rats, is bioavailable following oral gavage (F = 30%) and brain-penetrant (CSF conc/total plasma conc ratio = 0.8%). RO4491533 appeared to engage the central mGluR2 and mGluR3 receptors since the compound reversed the hypolocomotor effect of an mGluR2/3 orthosteric agonist LY379268 in a target-specific manner, as did the group II orthosteric mGluR2/3 antagonist LY341495. RO4491533 and LY341495 dose-dependently reduced immobility time of C57Bl6/J mice in the forced swim test. Also, RO4491533 and LY341495 were active in the tail suspension test in a line of Helpless (H) mice, a putative genetic model of depression. These data suggest that mGluR2/3 receptors are viable targets for development of novel pharmacotherapies for depression.

Structure-activity relationship of isoform selective inhibitors of Rac1/1b GTPase nucleotide binding.

The synthesis of a series of berberine, phenantridine and isoquinoline derivatives was realized to explore their Rho GTPase nucleotide inhibitory activity. The compounds were evaluated in a nucleotide binding competition assay against Rac1, Rac1b, Cdc42 and in a cellular Rac GTPase activation assay. The insertion of 19 AA in the splice variant Rac1b is shown to be sufficient to introduce a conformational difference that allows compounds 4, 21, 22, and 26 to exhibit selective inhibition of Rac 1b over Rac1.

Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues.

Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes.

Characterization of [(3)H]ucb 30889 binding to synaptic vesicle protein 2A in the rat spinal cord.

The novel antiepileptic drug levetiracetam ((2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA possesses a specific binding site in brain, which has very recently been identified as the synaptic vesicle protein SV 2 A. The aim of this study was to evaluate the presence of a levetiracetam binding site in the spinal cord and compare its properties to that in rat brain. We used [(3)H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide), a levetiracetam analogue, to perform binding assays, photoaffinity labelling and autoradiography experiments, and revealed the presence of SV 2 A by Western-blot analysis. [(3)H]ucb 30889 binding kinetics at 4 degrees C were biphasic and saturation binding curves were compatible with the labelling of a homogenous population of binding sites with a K(d) similar to that in brain. Competition curves with ligands known to interact with levetiracetam binding sites and photolabelling experiments indicated that [(3)H]ucb 30889 labels the same 90 kDa protein in both spinal cord and brain. Levetiracetam binding site was localised in the grey matter of the spinal cord and its expression was not modified in a model of neuropathic pain. This study demonstrates the presence of a specific levetiracetam binding site in the rat spinal cord, which is similar to that found in rat brain.

Activation of mitogen-activated protein kinase pathways during the death of PC12 cells is dependent on the state of differentiation.

PC12 cells that are differentiated with NGF and cAMP become totally dependent on these factors for their survival, unlike those that are differentiated with NGF alone. We have asked whether the MAP Kinases, ERKs, JNKs and p38s play a role in the cell death induced by withdrawal of trophic factors on NGF- and NGF/cAMP-differentiated PC12 cells. By Western-blot analyses with antibodies directed against the activated forms of these kinases, we show that when the trophic factors were withdrawn, ERK phosphorylation was reduced to very low levels within 1 h in both cases. Changes in the other enzymes were observed only in the NGF/cAMP-differentiated cells, in which the JNK phosphorylation increased about 160% by 6 h and that of p38 increased linearly to at least 18-fold throughout the cell death process. The increases in p38 and JNK phosphorylation were implicated in the death of the cells, since the p38 inhibitor PD169316 and the JNK inhibitor SP600125 were protective. These results demonstrate that the state of differentiation of PC12 cells, a model for the differentiation of sympathetic neurons, determines their vulnerability to cell death by modifying the state of phosphorylation and the regulation of specific kinases implicated in signal transduction pathways that are responsible for the survival or the death of these cells.

Mechanism based neurotoxicity of mGlu5 positive allosteric modulators--development challenges for a promising novel antipsychotic target.

Previous work has suggested that activation of mGlu5 receptor augments NMDA receptor function and thereby may constitute a rational approach addressing glutamate hypofunction in schizophrenia and a target for novel antipsychotic drug development. Here, we report the in vitro activity, in vivo efficacy and safety profile of 5PAM523 (4-Fluorophenyl){(2R,5S)-5-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-3-yl]-2-methylpiperidin-1-yl}methanone), a structurally novel positive allosteric modulator selective of mGlu5. In cells expressing human mGlu5 receptor, 5PAM523 potentiated threshold responses to glutamate in fluorometric calcium assays, but does not have any intrinsic agonist activity. 5PAM523 acts as an allosteric modulator as suggested by the binding studies showing that 5PAM523 did not displace the binding of the orthosteric ligand quisqualic acid, but did partially compete with the negative allosteric modulator, MPyEP. In vivo, 5PAM523 reversed amphetamine-induced locomotor activity in rats. Therefore, both the in vitro and in vivo data demonstrate that 5PAM523 acts as a selective mGlu5 PAM and exhibits anti-psychotic like activity. To study the potential for adverse effects and particularly neurotoxicity, brain histopathological exams were performed in rats treated for 4 days with 5PAM523 or vehicle. The brain exam revealed moderate to severe neuronal necrosis in the rats treated with the doses of 30 and 50 mg/kg, particularly in the auditory cortex and hippocampus. To investigate whether this neurotoxicity is mechanism specific to 5PAM523, similar safety studies were carried out with three other structurally distinct selective mGlu5 PAMs. Results revealed a comparable pattern of neuronal cell death. Finally, 5PAM523 was tested in mGlu5 knock-out (KO) and wild type (WT) mice. mGlu5 WT mice treated with 5PAM523 for 4 days at 100 mg/kg presented significant neuronal death in the auditory cortex and hippocampus. Conversely, mGlu5 KO mice did not show any neuronal loss by histopathology, suggesting that enhancement of mGlu5 function is responsible for the toxicity of 5PAM523. This study reveals for the first time that augmentation of mGlu5 function with selective allosteric modulators results in neurotoxicity.

Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death.

We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.

The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam.

Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of approximately 90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.