Reference limits for GAD65 and IA-2 autoantibodies by chemiluminescence immunoassay in Northern European adults and children.

The GAD65 and IA-2 antibodies (Abs) are biomarkers of the development of type 1 diabetes mellitus (T1DM) in both children and adults. The upper reference limit for the autoantibodies made by the manufacture was established on an adult Chinese population. Here, we established upper reference limits for Northern European adults and children in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Serum samples from healthy Danish children (0-18 years) and adults (18-70 years) were analysed for GAD65Ab and IA-2Ab using MAGLUMI 800 Chemiluminescence Immunoassay (CLIA). The Kruskal-Wallis test was used for evaluating differences between gender and age groups. No gender or age differences were found for neither GAD65Ab nor IA-2Ab, and a combined upper reference limit for both children and adults could be established. An upper reference limit of 5.1 IU/mL was defined for GAD65Ab and 11.5 U/mL for IA-2Ab. Our results showed a substantial discrepancy with the reference limits established by the manufacturer.

Fulminant onset of insulin-dependent diabetes with positive anti-GAD antibody titers during treatment with nivolumab in a patient with NSCLC.

Programmed cell death-1 (PD-1) and programmed cell death-ligand-1 (PD-L1) inhibitors have been highlighted in the field of cancer treatment. The interaction between PD-1 and PD-L1 is thought to play an important role in the regulation of the self-immune tolerance mechanism, so blocking these molecules may cause serious immune-related adverse events (IrAE), including fulminant insulin-dependent (type 1) diabetes. Here, we describe a patient with fulminant type 1 diabetes induced by nivolumab, an anti-PD-1 antibody. The patient, a 78-year-old man, was being treated with nivolumab as a third-line treatment for squamous cell carcinoma of the lung. After three cycles, he experienced an abrupt flare-up of the blood glucose within half a day. His blood glucose further increased without clinical symptoms until his hospital visit. Laboratory data showed the complete exhaustion of intrinsic insulin and the elevation of serum antibody titer to glutamic acid decarboxylase (GAD). Although the patient was previously diagnosed with non-insulin-dependent (type 2) diabetes, his disease activity had been well controlled with oral medication and low-dose insulin therapy until just before the flare-up. Because of the laboratory findings and the extremely rapid onset of hyperglycemia, a diagnosis of fulminant, rather than the rapid onset, type 1 diabetes related to nivolumab therapy was strongly suspected. Our case study indicates that fulminant hyperglycemia can occur extremely rapidly. The blood glucose of patients receiving PD-1 antibody therapy should be closely monitored.

Primary islet autoantibody at initial seroconversion and autoantibodies at diagnosis of type 1 diabetes as markers of disease heterogeneity.

OBJECTIVE: The relationship between patterns of islet autoantibodies at diagnosis and specificity of the first islet autoantibody at the initiation of autoimmunity was analyzed with the aim of identifying patterns informative of the primary autoantibodies. METHODS: Information about a single first autoantibody at seroconversion and autoantibody data at diagnosis were available for 128 children participating in the follow-up cohort of the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. Autoantibody data at diagnosis and genotyping results were also obtained from children in the Finnish Pediatric Diabetes Register (FPDR). RESULTS: Insulin autoantibodies (IAA) were the most common primary antibodies (N = 68), followed by those for glutamic acid decarboxylase (GADA; N = 38), IA-2 antigen (IA-2A; N = 13), and zinc transporter 8 (ZnT8A; N = 9), whereas at diagnosis, IA-2A were most frequent (N = 103), followed by IAA (N = 78), ZnT8A (N = 73), and GADA (N = 71). Accordingly, the presence of many specific autoantibodies at diagnosis was due to the secondary antibodies appearing after primary antibodies, and in some cases, the primary autoantibody, most often IAA, had already disappeared at the time of diagnosis. Many of the autoantibody combinations present at diagnosis could be assembled into groups associated with either IAA or GADA as first autoantibodies. These combinations, in children diagnosed below the age of 10 years in the FPDR, were found to be strongly associated with risk genotypes in either INS (IAA first) or IKZF4-ERBB3 (GADA first) genes. CONCLUSIONS: Autoantibody patterns at diagnosis may be informative on primary autoantibodies initiating autoimmunity in young children developing type 1 diabetes.

A novel approach in acidic disinfection through inhibition of acid resistance mechanisms; Maleic acid-mediated inhibition of glutamate decarboxylase activity enhances acid sensitivity of Listeria monocytogenes.

Here it is demonstrated a novel approach in disinfection regimes where specific molecular acid resistance systems are inhibited aiming to eliminate microorganisms under acidic conditions. Despite the importance of the Glutamate Decarboxylase (GAD) system for survival of Listeria monocytogenes and other pathogens under acidic conditions, its potential inhibition by specific compounds that could lead to its elimination from foods or food preparation premises has not been studied. The effects of maleic acid on the acid resistance of L. monocytogenes were investigated and found that it has a higher antimicrobial activity under acidic conditions than other organic acids, while this could not be explained by its pKa or Ka values. The effects were found to be more pronounced on strains with higher GAD activity. Maleic acid affected the extracellular GABA levels while it did not affect the intracellular ones. Maleic acid had a major impact mainly on GadD2 activity as also shown in cell lysates. Furthermore, it was demonstrated that maleic acid is able to partly remove biofilms of L. monocytogenes. Maleic acid is able to inhibit the GAD of L. monocytogenes significantly enhancing its sensitivity to acidic conditions and together with its ability to remove biofilms, make a good candidate for disinfection regimes.

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD-PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan).

The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of ß-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked ß-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.

Glutamic acid decarboxylase and islet antigen 2 antibody profiles in people with adult-onset diabetes mellitus: a comparison between mixed ethnic populations in Singapore and Germany.

AIM: To gain insight into the presence of islet cell autoimmunity in an ethnic Asian compared with a white European population. METHODS: For this cross-sectional study we recruited people with adult-onset diabetes (age of diagnosis 20-60 years), at tertiary referral centres in Germany (n=1020) and Singapore (n=1088). Glutamic acid decarboxylase and islet antigen 2 antibodies were measured according to Islet Autoantibody Standardization Program protocols. RESULTS: The prevalence of glutamic acid decarboxylase antibody positivity was 13.9% (95% CI 12.1-16.0; P<0.001) in the white European cohort compared with 6.8% (95% CI 5.5-8.4; P<0.001) in the Asian cohort. Glutamic acid decarboxylase antibody positivity was 11.4% (95% CI 7.7-16.6) in Indian, 6.0% (95% CI 3.6-9.9) in Malay and 5.8% (95% CI 4.3-7.7; P<0.001) in Chinese participants. In the white European participants, the prevalence of islet antigen 2 antibody positivity was 7.8% (95% CI 6.4-9.4) compared with 14.8% (95% CI 12.8-17.0; P<0.001) in the Asian cohort as a whole, and among the three ethnicities in the Asian cohort it was 12.4% (95% CI 8.6-17.7) in Indian, 16.8% (95% CI 12.6-22.2) in Malay and 15.7% (95% CI 13.2-18.6) in Chinese participants. Double antibody positivity was seen in 5.7% (95% CI 4.5-7.1) of white European participants compared with 1.6% (95% CI 1.0-2.5; P<0.01) of Asian participants. In the white European cohort, those who were glutamic acid decarboxylase autoantibody-positive had a lower BMI than those who were autoantibody-negative, but this trend was absent in the Asian cohort. CONCLUSIONS: A marked prevalence of islet cell autoimmunity was observed in people with adult-onset diabetes. While glutamic acid decarboxylase antibodies were more frequent in the European cohort, islet antigen 2 antibody positivity was highest in the three ethnic groups in Singapore, suggesting ethnic-specific differences in antibody profiles.

Validation of a rapid type 1 diabetes autoantibody screening assay for community-based screening of organ donors to identify subjects at increased risk for the disease.

The Network for Pancreatic Organ donors with Diabetes (nPOD) programme was developed in response to an unmet research need for human pancreatic tissue obtained from individuals with type 1 diabetes mellitus and people at increased risk [i.e. autoantibody (AAb)-positive] for the disease. This necessitated the establishment of a type 1 diabetes-specific AAb screening platform for organ procurement organizations (OPOs). Assay protocols for commercially available enzyme-linked immunosorbent assays (elisas) determining AAb against glutamic acid decarboxylase (GADA), insulinoma-associated protein-2 (IA-2A) and zinc transporter-8 (ZnT8A) were modified to identify AAb-positive donors within strict time requirements associated with organ donation programmes. These rapid elisas were evaluated by the international islet AAb standardization programme (IASP) and used by OPO laboratories as an adjunct to routine serological tests evaluating donors for organ transplantation. The rapid elisas performed well in three IASPs (2011, 2013, 2015) with 98-100% specificity for all three assays, including sensitivities of 64-82% (GADA), 60-64% (IA-2A) and 62-68% (ZnT8A). Since 2009, nPOD has screened 4442 organ donors by rapid elisa; 250 (5·6%) were identified as positive for one AAb and 14 (0.3%) for multiple AAb with 20 of these cases received by nPOD for follow-up studies (14 GADA+, two IA-2A(+) , four multiple AAb-positive). Rapid screening for type 1 diabetes-associated AAb in organ donors is feasible, allowing for identification of non-diabetic, high-risk individuals and procurement of valuable tissues for natural history studies of this disease.

Glutamic acid decarboxylase autoantibody-positivity post-partum is associated with impaired ß-cell function in women with gestational diabetes mellitus.

AIMS: To investigate whether the presence of glutamic acid decarboxylase (GAD) autoantibodies post-partum in women with prior gestational diabetes mellitus was associated with changes in metabolic characteristics, including ß-cell function and insulin sensitivity. METHODS: During 1997-2010, 407 women with gestational diabetes mellitus were offered a 3-month post-partum follow-up including anthropometrics, serum lipid profile, HbA1c and GAD autoantibodies, as well as a 2-h oral glucose tolerance test (OGTT) with blood glucose, serum insulin and C-peptide at 0, 30 and 120 min. Indices of insulin sensitivity and insulin secretion were estimated to assess insulin secretion adjusted for insulin sensitivity, disposition index (DI). RESULTS: Twenty-two (5.4%) women were positive for GAD autoantibodies (GAD+ve) and the remainder (94.6%) were negative for GAD autoantibodies (GAD-ve). The two groups had similar age and prevalence of diabetes mellitus. Women who were GAD+ve had significantly higher 2-h OGTT glucose concentrations during their index-pregnancy (10.5 vs. 9.8 mmol/l, P = 0.001), higher fasting glucose (5.2 vs. 5.0 mmol/l, P = 0.02) and higher 2-h glucose (7.8 vs. 7.1 mmol/l, P = 0.05) post-partum. Fasting levels of C-peptide and insulin were lower in GAD+ve women compared with GAD-ve women (520 vs. 761 pmol/l, P = 0.02 and 33 vs. 53 pmol/l, P = 0.05) Indices of insulin sensitivity were similar in GAD+ve and GAD-ve women, whereas all estimates of DI were significantly reduced in GAD+ve women. CONCLUSION: GAD+ve women had higher glucose levels and impaired insulin secretion adjusted for insulin sensitivity (DI) compared with GAD-ve women. The combination of OGTT and GAD autoantibodies post-partum identify women with impaired ß-cell function. These women should be followed with special focus on development of Type 1 diabetes.

A/H1N1 antibodies and TRIB2 autoantibodies in narcolepsy patients diagnosed in conjunction with the Pandemrix vaccination campaign in Sweden 2009-2010.

Narcolepsy is a lifelong sleep disorder related to hypocretin deficiency resulting from a specific loss of hypocretin-producing neurons in the lateral hypothalamic area. The disease is thought to be autoimmune due to a strong association with HLA-DQB1*06:02. In 2009 the World Health Organization (WHO) declared the H1N1 2009 flu pandemic (A/H1N1PDM09). In response to this, the Swedish vaccination campaign began in October of the same year, using the influenza vaccine Pandemrix(®). A few months later an excess of narcolepsy cases was observed. It is still unclear to what extent the vaccination campaign affected humoral autoimmunity associated with narcolepsy. We studied 47 patients with narcolepsy (6-69 years of age) and 80 healthy controls (3-61 years of age) selected after the Pandemrix vaccination campaign. The first aim was to determine antibodies against A/H1N1 and autoantibodies to Tribbles homolog 2 (TRIB2), a narcolepsy autoantigen candidate as well as to GAD65 and IA-2 as disease specificity controls. The second aim was to test if levels and frequencies of these antibodies and autoantibodies were associated with HLA-DQB1*06:02. In vitro transcribed and translated [(35)S]-methionine and -cysteine-labeled influenza A virus (A/California/04/2009/(H1N1)) segment 4 hemagglutinin was used to detect antibodies in a radiobinding assay. Autoantibodies to TRIB2, GAD65 and IA-2 were similarly detected in standard radiobinding assays. The narcolepsy patients had higher median levels of A/H1N1 antibodies than the controls (p = 0.006). A/H1N1 antibody levels were higher among the <13 years old (n = 12) compared to patients who were older than 30 years (n = 12, p = 0.014). Being HLA-DQB1*06:02 positive was associated with higher A/H1N1 antibody levels in both patients and controls (p = 0.026). Serum autoantibody levels to TRIB2 were low overall and high binders did not differ between patients and controls. We observed an association between levels of A/H1N1 antibodies and TRIB2 autoantibody levels particularly among the youngest narcolepsy patients (r = 0.819, p < 0.001). In conclusion, following the 2009 influenza pandemic vaccination, A/H1N1 antibody levels were associated with young age-at-onset narcolepsy patients positive for HLA-DQB1*06:02. The possibility that TRIB2 is an autoantigen in narcolepsy remains to be clarified. We could verify autoantibody responses against TRIB2 which needs to be determined in larger patient cohorts and control populations.

Similar weight-adjusted insulin secretion and insulin sensitivity in short-duration late autoimmune diabetes of adulthood (LADA) and type 2 diabetes: Action LADA 9 [corrected].

AIMS: To explore insulin sensitivity and insulin secretion in people with latent autoimmune diabetes in adulthood (LADA) compared with that in people with type 2 diabetes. METHODS: A total of 12 people with LADA, defined as glutamic acid decarboxylase (GAD) antibody positivity and > 1 year of insulin independency (group A) were age-matched pairwise to people with type 2 diabetes (group B) and to six people with type 2 diabetes of similar age and BMI (group C). ß-Cell function (first-phase insulin secretion and assessment of insulin pulsatility), insulin sensitivity (hyperinsulinemic-euglycemic clamp) and metabolic response during a mixed meal were studied. RESULTS: Both first-phase insulin secretion and insulin release during the meal were greater (P = 0.05 and P = 0.009, respectively) in type 2 diabetes as compared with LADA; these differences were lost on adjustment for BMI (group C) and could be explained by BMI alone in a multivariate analysis. Neither insulin pulsatility, incretin secretion nor insulin sensitivity differed among the groups. CONCLUSIONS: We found no evidence that LADA and type 2 diabetes were distinct disease entities beyond the differences explained by BMI.

Zinc transporter 8 autoantibody (ZnT8A) could help differentiate latent autoimmune diabetes in adults (LADA) from phenotypic type 2 diabetes mellitus.

BACKGROUND: The ZnT8A is an independent marker for diagnosis of type 1 diabetes mellitus. We investigated the distribution and clinical features of ZnT8A positive latent autoimmune diabetes in adult (LADA) patients to explore the potential diagnostic application. METHODS: A total of 3062 phenotypic T2DM patients were randomly selected from a national multicenter study - the LADA China Study. Radioligand binding assays were applied to detect the presence of ZnT8A, GADA and IA-2A. HbA1c , fasting C-peptide and serum lipid levels were followed up with ZnT8A positive patients. RESULTS: The positive prevalence of ZnT8A, GADA and IA-2A in phenotypic T2DM patients was 1.99% (61/3062), 6.43% (197/3062) and 1.96% (60/3062), respectively. The ZnT8A positivity was lower than that of GADA(x² = 74.8, p < 0.001) but was comparable with that of IA-2A (p > 0.05). The positivity of ZnT8A in IA-2A positive patients was higher than that in GADA positive patients (38.3% vs. 10.7%, x² = 24.8, p < 0.001). On the basis of GADA and IA-2A positivity, the ZnT8A assay enhanced the diagnostic prevalence of LADA from 7.58 to 8.62%. The LADA patients who were positive for ZnT8A had higher systolic blood pressure when compared with GADA positive cases (p = 0.049) and higher total cholesterol levels when compared with antibody-negative T2DM patients (p = 0.035). CONCLUSION: The detection of ZnT8A at the basis of GADA and IA-2A can improve diagnostic sensitivity of Chinese LADA.

Glutamate acid decarboxylase 1 promotes metastasis of human oral cancer by ß-catenin translocation and MMP7 activation.

BACKGROUND: Glutamate decarboxylase 1 (GAD1), a rate-limiting enzyme in the production of γ-aminobutyric acid (GABA), is found in the GABAergic neurons of the central nervous system. Little is known about the relevance of GAD1 to oral squamous cell carcinoma (OSCC). We investigated the expression status of GAD1 and its functional mechanisms in OSCCs. METHODS: We evaluated GAD1 mRNA and protein expressions in OSCC-derived cells using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. To assess the critical functions of GAD1, i.e., cellular proliferation, invasiveness, and migration, OSCC-derived cells were treated with the shRNA and specific GAD1 inhibitor, 3-mercaptopropionic acid (3-MPA). GAD1 expression in 80 patients with primary OSCCs was analyzed and compared to the clinicopathological behaviors of OSCC. RESULTS: qRT-PCR and immunoblotting analyses detected frequent up-regulation of GAD1 in OSCC-derived cells compared to human normal oral keratinocytes. Suppression of nuclear localization of ß-catenin and MMP7 secretion was observed in GAD1 knockdown and 3-MPA-treated cells. We also found low cellular invasiveness and migratory abilities in GAD1 knockdown and 3-MPA-treated cells. In the clinical samples, GAD1 expression in the primary OSCCs was significantly (P < 0.05) higher than in normal counterparts and was correlated significantly (P < 0.05) with regional lymph node metastasis. CONCLUSIONS: Our data showed that up-regulation of GAD1 was a characteristic event in OSCCs and that GAD1 was correlated with cellular invasiveness and migration by regulating ß-catenin translocation and MMP7 activation. GAD1 might play an important role in controlling tumoral invasiveness and metastasis in oral cancer.

Cerebellar fastigial nuclear GABAergic projections to the hypothalamus modulate immune function.

Our previous work has shown that the cerebellar fastigial nucleus (FN) is involved in modulation of lymphocyte function. Herein, we investigated effect of FN γ-aminobutyric acid (GABA)-ergic projections to the hypothalamus on lymphocytes to understand pathways and mechanisms underlying cerebellar immunomodulation. By injection of Texas red dextran amine (TRDA), an anterograde tracer, into FN, we found that the TRDA-labeled fibers from the FN traveled through the superior cerebellar peduncle (SCP), crossed in decussation of SCP (XSCP), entered the hypothalamus, and primarily terminated in the lateral hypothalamic area (LHA). Further, by injecting Fluoro-Ruby (FR), a retrograde tracer, in LHA, we observed that the FR-stained fibers retrogradely passed through XSCP and reached FN. Among these FR-positive neurons in the FN, there were GABA-immunoreactive cells. We then microinjected vigabatrin, which is an inhibitor of GABA-transaminase (GABA-T) that degrades GABA, bilaterally into FN. The vigabatrin treatment increased both number of GABA-immunoreactive neurons in FN-LHA projections and GABA content in the hypothalamus. Simultaneously, vigabatrin significantly reduced concanavalin A (Con A)-induced lymphocyte proliferation, anti-sheep red blood cell (SRBC) IgM antibody level, and natural killer (NK) cell number and cytotoxicity. In support of these findings, we inhibited GABA synthesis by using 3-mercaptopropionic acid (3-MP), which antagonizes glutamic acid decarboxylase (GAD). We found that the inhibition of GABA synthesis caused changes that were opposite to those when GABA was increased with vigabatrin. These findings show that the cerebellar FN has a direct GABAergic projection to the hypothalamus and that this projection actively participates in modulation of lymphocytes.

Estimation of ambient GABA levels in layer I of the mouse neonatal cortex in brain slices.

GABAergic synapses on Cajal-Retzius neurons in layer I of the murine neocortex experience GABA(B) receptor (GABA(B)R)-mediated tonic inhibition. Extracellular GABA concentration ([GABA](o)) that determines the strength of GABA(B)R-mediated inhibition is controlled by GABA transporters (GATs). In this study, we hypothesized that the strength of presynaptic GABA(B)R activation reflects [GABA](o) in the vicinity of synaptic contacts. Slices obtained from two age groups were used, namely postnatal days (P)2-3 and P5-7. GABAergic postsynaptic currents (IPSCs) were recorded using the whole-cell patch-clamp technique. Minimal electrical stimulation in layer I was applied to elicit evoked IPSCs (eIPSCs) using a paired-pulse protocol. Three parameters were selected for comparison: the mean eIPSC amplitude, paired-pulse ratio, and failure rate. When GAT-1 and GAT-2/3 were blocked by NO-711 (10 microM) and SNAP-5114 (40 microM), respectively, no tonic GABA(B)R-mediated inhibition was observed. In order to restore the control levels of GABA(B)R-mediated inhibition, 250 and 125 nm exogenous GABA was required at P2-3 and P5-7, respectively. Addition of 3-mercaptopropionic acid, a glutamate decarboxylase inhibitor, did not significantly change the obtained values arguing against the suggestion that a mechanism different from GATs contributes to [GABA](o) control. We conclude that juxtasynaptic [GABA](o) is higher (about 250 nM) at P2-3 than at P5-7 (about 125 nM). As both radial cell migration and corticogenesis in general are strongly dependent on [GABA](o) and the formation of the last layer 2/3 is finished by P4 in rodents, the observed [GABA](o) reduction in layer I might reflect this crucial event in the cortical development.

Initial report of γ-aminobutyric acidergic locomotion regulatory system and its 3-mercaptopropionic acid-sensitivity in metamorphic juvenile of sea urchin, Hemicentrotus pulcherrimus.

The γ-aminobutyric acid (GABA) signal transmission system (GSTS) contributes to larval swimming through the regulation of ciliary beating. However, whether this system also contributes to the primary podia (PP)-generated motility of juveniles remained unclear. The present study aimed to elucidate the involvement of the GSTS in the motility of metamorphic juveniles (juveniles) (1) by immunohistochemically elucidating the location of molecular constituents of the PP, and (2) by inhibiting the activity of GΑΒΑ decarboxylase (GAD) with 3-mercaptopropionic acid (3-MPA). During metamorphosis, the echinus rudiment protrudes its PP out of the body surface in 8-arm plutei. The PP expresses immunopositive signal (-IS) of GAD, GABA, GABAA receptor and tropomyosin, and is constituted with the GABA-IS negative distal tip and the GABA/GAD-IS gaiter region. The latter radiates distal projections to the disc that contains a GAD-IS cellular network. The juvenile body cavity houses a GABA/ßIII-tubulin-IS Penta-radial ring (PrR) that extends branches into each PP and several bridges to the GAD/GABA-IS Penta-radial plate (PrP) on the oral side but does not reach to the gaiter region. 3-MPA reversibly inhibits the juvenile motility and GABA-IS expression in the PrR/PrP complex. This indicates that the complex is the major contributor to the GABAergic motility in juveniles.

Clinical features and immunotherapy outcomes of anti-glutamic acid decarboxylase 65 antibody-associated neurological disorders.

We described the clinical and neuroimaging characteristics of seven Chinese patients with anti-GAD65 antibody-associated neurological disorders of whom epileptic seizures were the initial and main symptoms. All patients were given immunotherapy and followed up monthly. The outcome demonstrates that immunotherapy is helpful for non-seizure manifestations of anti-GAD65-associated neurological autoimmunity and is less effective in the treatment of seizures, yet partial responses can still occur in the early stage. Taken together we suggest a trial with immunotherapy in all patients in the early stage of the disease, and in patients with non-epilepsy symptoms in the later stage.

Investigation into the antimicrobial activity of fumarate against Listeria monocytogenes and its mode of action under acidic conditions.

Organic acids such as fumarate are commonly used as antimicrobials in foods. Apart from the classical mechanism of intracellular dissociation, weak acids are active through important additional mechanisms which are not well-defined. Fumarate, based on its low dissociation constants is expected to have a low antimicrobial activity which is not the case, suggesting additional antimicrobial effects. Previously, fumarate has been shown to inhibit the GAD system of E. coli and therefore, we investigated for first time how it affects this system in Listeria monocytogenes. We found that fumarate is highly antimicrobial towards L. monocytogenes under acidic conditions. We also show that in cell lysates and similarly to E. coli, fumarate inhibits the GAD system of L. monocytogenes. However, despite the inhibition and in contrast to E. coli, L. monocytogenes is able to counteract this and achieve a higher extracellular GAD output (measured by GABA export) in the presence of fumarate compared to its absence. The latter is achieved by a dramatic 9.44-fold increase in the transcription of gadD2 which is the main component of the extracellular GAD system. Interestingly, although maleate, the cis-isomer of fumarate results in a more dramatic 48.5-fold gadD2 upregulation than that of fumarate, the final GADe output is lower suggesting that maleate might be a stronger inhibitor of the GAD system. In contrast, the GADe removes more protons in the presence of fumarate than in the presence of HCl at the same pH. All the above suggest that there are additional effects by fumarate which might be associated with the intracellular GAD system (GADi) or other acid resistance systems. We assessed the GADi output by looking at the intracellular GABA pools which were not affected by fumarate. However, there are multiple pathways (e.g. GABA shunt) that can affect GABAi pools and we cannot conclusively suggest that GADi is affected. Furthermore, similarly to maleate, fumarate is able to eliminate L. monocytogenes in biofilms under acidic conditions. Overall, fumarate is a good candidate for L. monocytogenes decontamination and biofilm removal which is not toxic compared to the toxic maleate.

Effects of 4-methylimidazole on cerebral glutamate decarboxylase activity and specific GABA receptor binding in mice.

4-methylimidazole (4MeI) is a tremorogenic and convulsive agent of concern both in human and veterinary toxicology. The in vitro effects of 4MeI (5 microM-20 mM) on cerebral glutamate decarboxylase (GAD) activity and (in concentrations up to 50 mM) on binding of [(3)H]GABA to cerebral GABA receptors were tested in brain tissue from B6D2 mice. The effects of 1-methylimidazole (1MeI), 2-methylimidazole (2MeI), 4-methylhydroxy-imidazole (4MeOHI), imidazole-4-acetic acid (4AcI) (all in concentrations of 5-20 mM) and imidazole (20 mM) on GAD activity were also tested. In addition, the effect of a lethal dose of 4MeI (250 mg/kg ip) to B6D2 mice in vivo on the postmortem concentrations of gamma-aminobutyric acid (GABA) and glutamate in their brains were measured. In all experiments, student's t-test was used for statistical comparison. 4MeI in concentrations of 2 mM and above did inhibit GAD activity significantly in vitro, but glutamate and GABA concentrations in mouse brains after lethal 4MeI poisoning were not significantly different from control values. The effect of 2MeI on GAD activity was stronger than the effect of 4MeI. Binding of [(3)H]GABA to cerebral GABA receptors in vitro was significantly inhibited only at 4MeI concentrations of 5 mM and above. The results indicate that neither inhibition of GABA synthesis nor competitive inhibition of the binding of GABA to its receptors are likely mechanisms for the excitation and convulsions seen in 4MeI poisoning in animals.

Ascorbic Acid Supplementation Prevents the Detrimental Effects of Prenatal and Postnatal Lead Exposure on the Purkinje Cell and Related Proteins in the Cerebellum of Developing Rats.

We investigated the effects of lead (Pb) and ascorbic acid co-administration on rat cerebellar development. Prior to mating, rats were randomly divided into control, Pb, and Pb plus ascorbic acid (PA) groups. Pregnant rats were administered Pb in drinking water (0.3% Pb acetate), and ascorbic acid (100 mg/kg) via oral intubation until the end of the experiment. Offspring were sacrificed at postnatal day 21, the age at which the morphology of the cerebellar cortex in developing pups is similar to that of the adult brain. In the cerebellum, Pb exposure significantly reduced Purkinje cells and ascorbic acid prevented their reduction. Along with the change of the Purkinje cells, long-term Pb exposure significantly reduced the expression of the synaptic marker (synaptophysin), γ-aminobutyric acid (GABA)-synthesizing enzyme (glutamic acid decarboxylase 67), and axonal myelin basic protein while ascorbic acid co-treatment attenuated Pb-mediated reduction of these proteins in the cerebellum of pups. However, glutamatergic N-methyl-D-aspartate receptor subtype 1 (NMDAR1), anchoring postsynaptic density protein 95 (PSD95), and antioxidant superoxide dismutases (SODs) were adversely changed; Pb exposure increased the expression of NMDAR1, PSD95, and SODs while ascorbic acid co-administration attenuated Pb-mediated induction. Although further studies are required about the neurotoxicity of the Pb exposure, the results presented here suggest that developmental Pb exposure disrupted normal development of Purkinje cells by increasing glutamatergic and oxidative stress in the cerebellum. Additionally, ascorbic acid co-treatment is beneficial in attenuating prenatal and postnatal Pb exposure-induced maldevelopment of Purkinje cells in the developing cerebellum.

Isoniazid-induced reduction in GABAergic neurotransmission alters the function of the cerebellar cortical circuit.

The cerebellar cortex contributes to the control of movement, coordination, and certain cognitive functions. The cerebellar network is composed of five different types of neurons that are wired together in a repetitive module. Given that four of these five neurons synthesize and release GABA, this inhibitory neurotransmitter plays a central role in regulation of the excitability and correct functioning of the cerebellar cortex. We have now used isoniazid, an inhibitor of glutamic acid decarboxylase, the enzyme responsible for the synthesis of GABA, to evaluate the contribution of GABAergic transmission in different types of cerebellar cortical neurons to the functioning of the cerebellar circuit. Parasagittal cerebellar slices were prepared from 28- to 40-day-old male rats and were subjected to patch-clamp recording in the voltage- or current-clamp mode. Exposure of the tissue slices to isoniazid (10 mM) resulted in a decrease in the level of GABAergic transmission in Purkinje cells and a consequent increase in the firing rate of spontaneous action potentials that was apparent after 40 min. In granule neurons, isoniazid reduced both tonic and phasic GABAergic currents and thereby altered the flow of information across the cerebellar cortex. Our data support the notion that the amount of GABA at the synaptic level is a major determinant of the excitability of the cerebellar cortex, and they suggest that isoniazid may be a useful tool with which to study the function of the cerebellar network.