Cladribine modifies functional properties of microglia.

Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. We examined if CdA modifies the phenotype and function of naive and activated primary mouse microglia, when applied in the concentrations 0·1-1 µM that putatively overlap human cerebrospinal fluid (CSF) concentrations. Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti-inflammatory interleukin (IL)-4 were treated with different concentrations of CdA for 24 h. Viability was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Phagocytotic ability and morphology were examined by flow cytometry and random migration using IncuCyte Zoom and TrackMate. Change in gene expression was examined by quantitative polymerase chain reaction (qPCR) and protein secretion by Meso Scale Discovery. We found that LPS and IL-4 up-regulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0·1-1 µM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 µM increased the IL-4-induced gene expression of arginase 1 (Arg1) and LPS-induced expression of IL-1ß, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS) and Arg1, but protein secretion remained unaffected. CdA 10 µM potentiated the increased expression of anti-inflammatory TNF receptor 2 (TNF-R2) but not TNF-R1 induced by LPS. This suggests that microglia acquire a less activated phenotype when treated with 0·1-1 µM CdA that putatively overlaps human CSF concentrations. This may be related to the up-regulated gene expression of DCK upon activation, and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.

Autoimmune encephalitis associated with voltage-gated potassium channels-complex and leucine-rich glioma-inactivated 1 antibodies - a national cohort study.

BACKGROUND AND PURPOSE: The aim of this study was to describe clinical and paraclinical characteristics of all Danish patients who tested positive for anti-voltage-gated potassium channels (VGKC)-complex, anti-leucine-rich glioma-inactivated 1 (LGI1) and anti-contactin-associated protein-2 antibodies in the serum/cerebrospinal fluid between 2009 and 2013 with follow-up interviews in 2015 and 2016. METHODS: We evaluated antibody status, symptoms leading to testing, course of disease, suspected diagnosis and time of admission as well as diagnosis and treatment. All magnetic resonance imaging, electroencephalography and 18 F-fluorodeoxyglucose positron emission tomography scans were re-evaluated by experts in the field. RESULTS: A total of 28/192 patients tested positive for VGKC-complex antibodies by radioimmunoassay and indirect immunofluorescence; 17 had antibodies to LGI1 and 6/7 of the available cerebrospinal fluids from these patients were seropositive. These 17 patients all had a clinical phenotype appropriate to LGI1 antibodies. The remaining 11 were LGI1 negative (n = 4) or not tested (n = 7). Of these, two had a phenotype consistent with limbic encephalitis. The remaining phenotypes were Guillain-Barré syndrome, Creutzfeldt-Jakob disease, neuromyotonia and anti-N-methyl-D-aspartate receptor encephalitis. Magnetic resonance imaging abnormalities were demonstrated in 69% of the LGI1-positive patients. Two patients with normal magnetic resonance imaging demonstrated temporal lobe hypermetabolism using 18 F-fluorodeoxyglucose positron emission tomography. Abnormal electroencephalography recordings were found in 86% of the patients. Upon follow-up (median 3.2 years), the median modified Rankin Scale score of anti-LGI1-positive patients was 2 and only two patients reported seizures in the past year. CONCLUSIONS: Patients diagnosed with anti-LGI1 autoimmune encephalitis increased significantly from 2009 to 2014, probably due to increased awareness. In contrast to seropositive anti-VGKC-complex patients, all anti-LGI1-positive patients presented with a classical limbic encephalitis. The majority of patients recovered well.

Identification of a novel HLA-B allele, HLA-B*08:177, in a Hungarian patient and her HLA identical sibling.

The newly detected HLA-B*08:177 differs from HLA-B*08:01:01:01 by 1 single nucleotide substitution at position 365 of exon 3.

l-Arginine Pathway Metabolites Predict Need for Intra-operative Shunt During Carotid Endarterectomy.

OBJECTIVE/BACKGROUND: Asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) synthesis and is a marker of atherosclerosis. This study examined the correlation between pre-operative l-arginine and ADMA concentration during carotid endarterectomy (CEA), and jugular lactate indicating anaerobic cerebral metabolism, jugular S100B reflecting blood-brain barrier integrity, and with factors of surgical intervention. METHODS: The concentration of l-arginine, ADMA, and symmetric dimethylarginine was measured in blood taken under regional anaesthesia from the radial artery of 55 patients prior to CEA. Blood gas parameters, concentration of lactate, and S100B were also serially measured in blood taken from both the radial artery and the jugular bulb before and after carotid clamping, and after release of the clamp. To estimate anaerobic metabolism, the jugulo-arterial ratio of CO2 gap/oxygen extraction was calculated. RESULTS: Positive correlation was found between pre-operative ADMA levels and the ratio of jugulo-arterial CO2 gap/oxygen extraction during clamp and reperfusion (p = .005 and p = .01, respectively). An inverse correlation was found between the pre-operative l-arginine concentration and jugular lactate at each time point (both p = .002). The critical pre-operative level of l-arginine was determined by receiver operator curve analysis. If l-arginine was below the cutoff value of 35 µmol/L, jugular S100B concentration was higher 24 h post-operatively (p = .03), and jugular lactate levels were increased during reperfusion (p = .02). The median pre-operative concentration of l-arginine was lower in patients requiring an intra-operative shunt than in patients without need of shunt (median: 30.3 µmol/L [interquartile range 24.4-34.4 µmol/L] vs. 57.6 µmol/L [interquartile range 42.3-74.5 µmol/L]; p = .002). CONCLUSION: High pre-operative ADMA concentration predicts poor cerebral perfusion indicated by elevated jugulo-arterial CO2 gap/oxygen extraction. Low pre-operative l-arginine concentration predicts the need for a shunt. The inverse correlation between pre-operative l-arginine concentration and both jugular lactate and S100B during carotid clamping suggests a protective role of the NO donor l-arginine.

HLA genetic diversity in Hungarians and Hungarian Gypsies: complementary differentiation patterns and demographic signals revealed by HLA-A, -B and -DRB1 in Central Europe.

Systematic analyses of human leukocyte antigen (HLA) profiles in different populations may increase the efficiency of bone marrow donor selection and help reconstructing human peopling history. We typed HLA-A, -B, and -DRB1 allele groups in two bone marrow donor cohorts of 2402 Hungarians and 186 Hungarian Gypsies and compared them with several Central-European, Spanish Gypsy, and Indian populations. Our results indicate that different European Gypsy populations share a common origin but diverged genetically as a consequence of founder effect and rapid genetic drift, whereas other European populations are related genetically in relation to geography. This study also suggests that while HLA-A accurately depicts the effects of genetic drift, HLA-B, and -DRB1 conserve more signatures of ancient population relationships, as a result of balancing selection.

Low occurrence of the HLA-C*04:09N allele in a large Hungarian cohort.

The presence of null alleles may affect the outcome of stem cell transplantation. HLA-C*04:09N was defined as 'common' with a frequency of 2-5/1000 in Caucasians, and its presence is routinely tested as part of haplotypes HLA-A*02:01/A*23:01-B*44:03-DRB1*07:01-DQB1*02:01. We aimed to investigate HLA-C*04:09N in a representative Hungarian cohort. HLA-typing data of 7345 unrelated persons were analyzed. The presence of HLA-C*04:09N was excluded in 157 chromosomes with either serology typing or with an allele-specific polymerase chain reaction for HLA-C*04:09N. HLA-C*04:09N was identified in a single chromosome with HLA-A*02, B*44, C*04, DRB1*07 resulting in a HLA-C*04:09N allele frequency of 0.0068% (1/14,690). This is approximately a 10- to 40-fold lower frequency compared with the previous data. Our results emphasize the need of precise local population-specific HLA-data, allowing appropriate modifications of local HLA-typing protocols.

High anti-EBNA-1 IgG levels are associated with early-onset myasthenia gravis.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acethylcholine receptor (AchR) of the neuromuscular junction in the majority of patients. METHODS: Here, we examined IgG antibodies against the type 1 nuclear antigen of Epstein-Barr virus (EBNA-1) in the sera of 158 patients with MG compared to 184 healthy controls. RESULTS: Although serum concentration in the sera was not different, high anti-EBNA-1 IgG titers (above 90th percentile of the normal values) were more common in the patients (26.6 vs. 16.3%, P=0.024). In addition, high EBNA-1 IgG levels occurred more frequently amongst the 94 patients with early-onset myasthenia gravis (EOMG, 30.8%) as compared to the 64 patients with late-onset disease (LOMG, 14.1%) (P=0.021). Using multiple logistic regression, high serum concentration of the anti-EBNA-1 IgG antibodies was significantly associated with EOMG (OR: 3.17, P=0.027), even after adjustment for sex, presence/absence of anti-AchR antibodies and presence/absence of anti-Titin antibodies. Out of 39 patients with EOMG, who underwent thymectomy, 18 patients (46%) had thymoma, 6 had thymic hyperplasia (15%), and 15 patients had thymic atrophy (39%); there was no difference comparing EBNA-1 antibody titers in the sera. As no correlation was found between the titers of anti-AchR, anti-Titin, and EBNA-1 antibodies, a dysregulated heterogeneous B-cell response was unlikely to be responsible for the elevated levels of EBV-associated antibody in patients. CONCLUSIONS: In summary, our data suggest that high levels of EBNA-1 antibodies are more common in MG compared to healthy controls and are especially associated with EOMG.

Long-term effects of combined immunosuppressive treatment on myasthenic crisis.

BACKGROUND AND PURPOSE: We studied the long-term crisis-preventing effect of combined prednisolone-azathioprine (PR-AZA) treatment in myasthenia gravis (MG). METHODS: Case-control study with a median follow-up of 64 months in the treated group, and 80 months in the non-treated group. Sixty-nine patients with episodes of myasthenic crisis (MC) were treated and followed prospectively in 1990-2004. Twenty-seven patients had MC between 1990 and 1996, and were not treated with immunosuppressants on long-term. Long-term PR-AZA treatment was introduced in another 42 patients, who developed MC in 1997-2004. The difference in the frequency of repeated MCs between the treated and the non-treated group during long-term follow-up was investigated. As secondary end-points, we analyzed the number of admittances to the ICU; the number of mechanical ventilation episodes; the duration of ICU treatment; the characteristics of the applied treatment; and the functional outcome of the patients 1 month after the onset of the crisis. RESULTS: Recurrent MCs occurred in 74% of the non-treated and 19% of the treated group (P < 0.001). The number of ICU admissions (P = 0.005) and mechanical ventilation events (P = 0.002) were also reduced. The highly significant MC preventing effect of PR-AZA was evident after 6 months. CONCLUSIONS: After the initial 6 months of therapy, PR-AZA is effective in preventing MC.