Routine diagnostics for neural antibodies,clinical correlates,treatment and functional outcome

Objective

To determine frequencies, interlaboratory reproducibility, clinical ratings, and prognostic implications of neural antibodies in a routine laboratory setting in patients with suspected neuropsychiatric autoimmune conditions.

Methods

Earliest available samples from 10,919 patients were tested for a broad panel of neural antibodies. Sera that reacted with leucine-rich glioma-inactivated protein 1 (LGI1), contactin-associated protein-2 (CASPR2), or the voltage-gated potassium channel (VGKC) complex were retested for LGI1 and CASPR2 antibodies by another laboratory. Physicians in charge of patients with positive antibody results retrospectively reported on clinical, treatment, and outcome parameters.

Results

Positive results were obtained for 576 patients (5.3%). Median disease duration was 6 months (interquartile range 0.6–46 months). In most patients, antibodies were detected both in CSF and serum. However, in 16 (28%) patients with N-methyl-d-aspartate receptor (NMDAR) antibodies, this diagnosis could be made only in cerebrospinal fluid (CSF). The two laboratories agreed largely on LGI1 and CASPR2 antibody diagnoses (κ = 0.95). The clinicians (413 responses, 71.7%) rated two-thirds of the antibody-positive patients as autoimmune. Antibodies against the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), NMDAR (CSF or high serum titer), γ-aminobutyric acid-B receptor (GABABR), and LGI1 had ≥ 90% positive ratings, whereas antibodies against the glycine receptor, VGKC complex, or otherwise unspecified neuropil had ≤ 40% positive ratings. Of the patients with surface antibodies, 64% improved after ≥ 3 months, mostly with ≥ 1 immunotherapy intervention.

Conclusions

This novel approach starting from routine diagnostics in a dedicated laboratory provides reliable and useful results with therapeutic implications. Counseling should consider clinical presentation, demographic features, and antibody titers of the individual patient.

     

Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study.

Two commercially available drug-screening assays were evaluated: the Roche kinetic interaction of microparticles in solution (KIMS) assay and the Microgenics cloned enzyme donor immunoassay (CEDIA). Urine samples from known drug-abuse patients were analyzed for amphetamines, barbiturates, benzodiazepines, benzoylecgonine, cannabinoids, LSD, methadone and opiates. Samples with discordant findings for the two assays were analyzed by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/electron capture detection (GC/ECD). Amphetamines showed 96.0% concordant results, with two false positive findings by CEDIA, three by KIMS and a further two false negatives by KIMS. Barbiturates showed 99.4% concordant results, with one false negative by KIMS. Benzodiazepines showed 97.4% concordant results, with two false negatives by KIMS (cutoff 100 microg/L, CEDIA cutoff 300 microg/L). Benzoylecgonine showed 17.8% concordant positive and 82.2% concordant negative results and no false finding by either assay. Cannabinoids showed 99.3% concordant results, with one sample negative by KIMS at a cutoff of 50 microg/L and positive by CEDIA (cutoff 25 microg/L). For LSD, 6.7% of findings were not in agreement. Methadone showed 97.5% concordant results, with two false positives by CEDIA, and one false positive and one false negative by KIMS. Opiates showed 96.9% concordant results, with no false KIMS results, but four false positives by CEDIA. The results indicate that the agreement of the CEDIA and KIMS results for the eight drugs is rather good (93.3-100%).     

Intracellular alkalinization causes Mg2+ release from intracellular binding sites in leech Retzius neurones

 Four-barrelled ion-sensitive microelectrodes were developed to enable simultaneous measurement of intracellular free Mg²⁺ and Na⁺ concentrations ([Mg²⁺]_i, [Na⁺]_i), intracellular pH and the membrane potential. The electrodes were used to investigate pH-induced [Mg²⁺]_i changes in Retzius neurones of the leech Hirudo medicinalis. The application of propionate or CO₂/HCO₃ ^–-buffered bath solutions caused a transient intracellular acidification, an initial [Mg²⁺]_i decrease and a continuous [Na⁺]_i increase. In the presence of CO₂/HCO₃ ^– this [Na⁺]_i increase was more pronounced and might be the reason for the slow increase in [Mg²⁺]_i following the initial decrease. The withdrawal of propionate or CO₂/HCO₃ ^–-buffered bath solutions caused a transient alkalinization which was accompanied by a slight but significant [Mg²⁺]_i increase, even in the nominal absence of extracellular Mg²⁺, while [Na⁺]_i returned to its original value. The alkalinization-induced [Mg²⁺]_i increase could be reduced to about 50% by the application of 1–10 μM cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (MTP). Phenylarsine oxide, an MTP activator, caused a [Mg²⁺]_i increase with characteristics similar to those of the alkalinization-induced increase, which could not be attributed to any changes in [Na⁺]_i or pH_i. It is concluded that an intracellular alkalinization might induce the release of Mg²⁺ from intracellular stores. Received: 8 May 1997 / Received after revision: 31 July 1997 / Accepted: 19 August 1997     

The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.     

A neuroprotective function for the hematopoietic protein granulocyte-macrophage colony stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine responsible for the proliferation, differentiation, and maturation of cells of the myeloid lineage, which was cloned more than 20 years ago. Here we uncovered a novel function of GM-CSF in the central nervous system (CNS). We identified the GM-CSF alpha-receptor as an upregulated gene in a screen for ischemia-induced genes in the cortex. This receptor is broadly expressed on neurons throughout the brain together with its ligand and induced by ischemic insults. In primary cortical neurons and human neuroblastoma cells, GM-CSF counteracts programmed cell death and induces BCL-2 and BCL-Xl expression in a dose- and time-dependent manner. Of the signaling pathways studied, GM-CSF most prominently induced the PI3K-Akt pathway, and inhibition of Akt strongly decreased antiapoptotic activity. Intravenously given GM-CSF passes the blood-brain barrier, and decreases infarct damage in two different experimental stroke models (middle cerebral artery occlusion (MCAO), and combined common carotid/distal MCA occlusion) concomitant with induction of BCL-Xl expression. Thus, GM-CSF acts as a neuroprotective protein in the CNS. This finding is remarkably reminiscent of the recently discovered functionality of two other hematopoietic factors, erythropoietin and granulocyte colony-stimulating factor in the CNS. The identification of a third hematopoietic factor acting as a neurotrophic factor in the CNS suggests a common principle in the functional evolution of these factors. Clinically, GM-CSF now broadens the repertoire of hematopoietic factors available as novel drug candidates for stroke and neurodegenerative diseases.     

Mg2+-malate co-transport, a mechanism for Na+-independent Mg2+ transport in neurons of the leech Hirudo medicinalis

Mg2+-extrusion from Mg2+-loaded neurons of the leech, Hirudo medicinalis, is mediated mainly by Na+/Mg2+ antiport. However, in a number of leech neurons, Mg2+ is extruded in the nominal absence of extracellular Na+, indicating the existence of an additional, Na+-independent Mg2+ transport mechanism. This mechanism was investigated using electrophysiological and microfluorimetrical techniques. The rate of Na+-independent Mg2+ extrusion from Mg2+-loaded leech neurons was found to be independent of extracellular Ca2+, K+, NO3-, HCO3-, SO4(2-), HPO4(2-), and of intra- and extracellular pH. Na+-independent Mg2+ extrusion was not inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), furosemide, ouabain, vanadate, iodoacetate, 4-amino-hippurate, or alpha-cyano-4-hydroxycinnamate and was not influenced by changes in the membrane potential in voltage-clamp experiments. Na+-independent Mg2+ extrusion was, however, inhibited by the application of 2 mM probenecid, a blocker of organic anion transporters, suggesting that Mg2+ might be co-transported with organic anions. Extracellularly, of all organic anions tested (malate, citrate, lactate, alpha-ketoglutarate, and 4-amino-hippurate) only high, but physiological, concentrations of malate (30 mM) had a significant inhibitory effect on Na+-independent Mg2+ extrusion. Intracellularly, iontophoretically injected malate, citrate, or fura-2, but not Cl-, alpha-ketoglutarate, glutamate, succinate, or urate, were stimulating Na+-independent Mg2+ extrusion from those neurons that initially did not extrude Mg2+ in Na+-free solutions. Our data indicate that Mg2+ is co-transported with organic anions, preferably with malate, the predominant extracellular anion in the leech. The proposed model implies that, under experimental conditions, malate drives Mg2+ extrusion, whereas under physiological conditions, malate is actively taken up, driven by Mg2+, so that malate can be metabolized.     

Voltage-dependent Ca influx into identified leech neurones

We determined the relationships between the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential (E_m) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca²⁺]_i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K⁺ concentration ([K⁺]_o) up to 89 mM caused a persistent increase in [Ca²⁺]_i, which was abolished in Ca²⁺-free solution indicating that it was due to Ca²⁺ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca²⁺]_i increase ranged between −40 and −25 mV. The different threshold potentials as well as differences in the relationships between [Ca²⁺]_i and E_m were partly due to the cell-specific generation of action potentials. In Na⁺-free solution, the action potentials were suppressed and the [Ca²⁺]_i/E_m relationships were similar. The K⁺-induced [Ca²⁺]_i increase was inhibited by the polyvalent cations Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, and La³⁺, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K⁺-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca²⁺ channels with similar properties.