Repeated social defeat stress exacerbates lipopolysaccharide-induced behavioural deficits in mice: ameliorative role of Chrysophyllum albidum fruit extract through anti-neuroinflammation, antioxidant and neurochemical balance.

Development of neuropsychiatric disorder is associated with stress-related increase in pro-inflammatory cytokines. Chrysophyllum albidum fruit is an edible tropical fruit containing vitamins and phenolic compounds, well known for their anti-inflammatory and antioxidant activities. This study was designed to investigate the neuroprotective effect of C. albidum fruit extract (CAFE) on stress and lipopolysaccharide (LPS)-induced behavioral and neurochemical impairments in mice. Male Swiss mice were divided into 6 groups (n = 6). Groups 1-3 were orally treated daily for 14 days with normal saline (0.1 mL/10 g), CAFE (100 mg/kg) and Ferulic acid (FA, 10 mg/kg), and left in home cage as controls. Groups 4-6 were treated similarly but subjected to repeated social defeat (RSD) stress using the resident-intruder model from days 1-14. The RSD-animals were injected with LPS (125 µg/kg, i.p) 60 min after each RSD session from days 8-14. Neurobehavioral functions: locomotor, cognitive and anxiety-like behaviors were assessed 24 h after the last treatment. Pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α), dopamine, acetylcholinesterase, glutamic acid decarboxylase (GAD), malondialdehyde, nitrites, and reduced glutathione (GSH) were determined in brain tissue. CAFE significantly attenuated RSD and LPS-induced hypolocomotion, cognitive impairment and anxiety-like behavior when compared to the control. Treatment with CAFE also significantly reversed the negative effects of RSD and LPS on pro-inflammatory cytokines, dopamine, acetylcholinesterase, GAD, and oxidative-nitrosative stress levels. The findings clearly indicated that Chrysophyllum albidum fruit demonstrated neuroprotective effects and can play a key role in mitigating against chronic stress and inflammation linked to neuropsychiatric disorders.

Effect of Immune Activation during Early Gestation or Late Gestation on Inhibitory Markers in Adult Male Rats.

People with schizophrenia exhibit deficits in inhibitory neurons and cognition. The timing of maternal immune activation (MIA) may present distinct schizophrenia-like phenotypes in progeny. We investigated whether early gestation [gestational day (GD) 10] or late gestation (GD19) MIA, via viral mimetic polyI:C, produces deficits in inhibitory neuron indices (GAD1, PVALB, SST, SSTR2 mRNAs) within cortical, striatal, and hippocampal subregions of male adult rat offspring. In situ hybridisation revealed that polyI:C offspring had: (1) SST mRNA reductions in the cingulate cortex and nucleus accumbens shell, regardless of MIA timing; (2) SSTR2 mRNA reductions in the cortex and striatum of GD19, but not GD10, MIA; (3) no alterations in cortical or striatal GAD1 mRNA of polyI:C offspring, but an expected reduction of PVALB mRNA in the infralimbic cortex, and; (4) no alterations in inhibitory markers in hippocampus. Maternal IL-6 response negatively correlated with adult offspring SST mRNA in cortex and striatum, but not hippocampus. These results show lasting inhibitory-related deficits in cortex and striatum in adult offspring from MIA. SST downregulation in specific cortical and striatal subregions, with additional deficits in somatostatin-related signalling through SSTR2, may contribute to some of the adult behavioural changes resulting from MIA and its timing.

Estrogen Treatment Reverses Prematurity-Induced Disruption in Cortical Interneuron Population.

Development of cortical interneurons continues until the end of human pregnancy. Premature birth deprives the newborns from the supply of maternal estrogen and a secure intrauterine environment. Indeed, preterm infants suffer from neurobehavioral disorders. This can result from both preterm birth and associated postnatal complications, which might disrupt recruitment and maturation of cortical interneurons. We hypothesized that interneuron subtypes, including parvalbumin-positive (PV+), somatostatin-positive (SST+), calretinin-positive (CalR+), and neuropeptide Y-positive (NPY+) interneurons, were recruited in the upper and lower cortical layers in a distinct manner with advancing gestational age. In addition, preterm birth would disrupt the heterogeneity of cortical interneurons, which might be reversed by estrogen treatment. These hypotheses were tested by analyzing autopsy samples from premature infants and evaluating the effect of estrogen supplementation in prematurely delivered rabbits. The PV+ and CalR+ neurons were abundant, whereas SST+ and NPY+ neurons were few in cortical layers of preterm human infants. Premature birth of infants reduced the density of PV+ or GAD67+ neurons and increased SST+ interneurons in the upper cortical layers. Importantly, 17 ß-estradiol treatment in preterm rabbits increased the number of PV+ neurons in the upper cortical layers relative to controls at postnatal day 14 (P14) and P21 and transiently reduced SST population at P14. Moreover, protein and mRNA levels of Arx, a key regulator of cortical interneuron maturation and migration, were higher in estrogen-treated rabbits relative to controls. Therefore, deficits in PV+ and excess of SST+ neurons in premature newborns are ameliorated by estrogen replacement, which can be attributed to elevated Arx levels. Estrogen replacement might enhance neurodevelopmental outcomes in extremely preterm infants.SIGNIFICANCE STATEMENT Premature birth often leads to neurodevelopmental delays and behavioral disorders, which may be ascribed to disturbances in the development and maturation of cortical interneurons. Here, we show that preterm birth in humans is associated with reduced population of parvalbumin-positive (PV+) neurons and an excess of somatostatin-expressing interneurons in the cerebral cortex. More importantly, 17 ß-estradiol treatment increased the number of PV+ neurons in preterm-born rabbits, which appears to be mediated by an elevation in the expression of Arx transcription factor. Hence the present study highlights prematurity-induced reduction in PV+ neurons in human infants and reversal in their population by estrogen replacement in preterm rabbits. Because preterm birth drops plasma estrogen level 100-fold, estrogen replacement in extremely preterm infants might improve their developmental outcome and minimize neurobehavioral disorders.

Marcadores de inflamación y autoinmunidad en pacientes con diabetes tipo 1 / And inflammation markers in patients with diabetes autoimmunity type 1

To determine the serological levels of inflammatory markers and autoimmunity in patients with T1D compared with controls, and determined its relation to the duration of diabetes. Methods: We selected 139 patients with T1D without chronic complications of diabetes, and 110 control subjects without family history of diabetes. Serological ultrasensitive C-reactive protein levels (usCRP), interleukin- 6 and adhesion protein VCAM through ELISA assay were determined. Autoimmune profile was also analyzed through GAD65, IA-2 and ZnT8 autoantibodies. Results: Increased levels of usCRP 1.74 (0.10 to 13.6) vs 1.08 (0.40 to 3.70) ng/ml (p < 0.03), VCAM 236.0 (122.2 to 693.5) vs 185.4 (101.3 to 421.3) ng/ml, p < 0.02 and IL-6 1.73 (0.40 to 9.10) vs 1.28 (0.30 to 4.60) ng/ml, p < 0.05 was found in the group of T1D patients compared with the control group. When analyzing inflammatory markers according to age groups (0-10 years and > 10 years), the values of usCRP were higher in the second group. There was no significant association between patients with DM1 and autoimmune positive profile with a higher frequency of markers of inflammation. Conclusions: These results suggest the presence of pro-inflammatory state is considerably more frequent in patients with T1D. The increased level of usCRP and IL -6 and according to age of the patients could indicate a possible role of adiposity and weight gain during the adolescence in the higher frequency of inflammatory markers in T1D patients...

Submerged fermentation of Lactobacillus rhamnosus YS9 for γ aminobutyric acid (GABA) production

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in central nervous system, and its application in drugs and functional foods has attracted great attention. To enhance production of y-aminobutyric acid, Lactobacillus rhamnosus YS9, a strain isolated from Chinese traditional fermented food pickled vegetable, was grown under submerged fermentation. Its cultivation conditions were investigated. When culture pH condition was adjusted to the optimal pH of glutamate decarboxylase activity, culture of Lb. rhamnosus YS9 in medium supplemented with 200 mM of monosodium glutamate and 200 µM of pyridoxal phosphate (PLP), produced 187 mM of GABA.

Production of gaba (γ - aminobutyric acid) by microorganisms: a review

GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods.

Imbalance of interneuron distribution between neocortex and basal ganglia: consideration of epileptogenesis of focal cortical dysplasia.

AIM: The balance of excitation and inhibition of neurons and neuronal network is very important to perform complete neuronal function. Damage or loss of inhibitory γ-aminobutyric acid (GABA)-ergic interneuron is associated with impaired inhibitory control of cortical pyramidal neurons, leading to hyperexcitability and epileptogenesis. Ectopic neurons in the basal ganglia are to be one of the pathological features of epileptogenesis. In the present study, we investigated distribution of interneuron subtypes between neocortex and caudate nucleus. METHODS: We performed immunohistochemistry of GABA, glutamic acid decarboxylase (GAD), calretinin (CR), calbindin (CB), parvalbumin (PV) and neuropeptide. We used surgical materials of four focal cortical dysplasia (FCD) cases, having lesions of neocortex and caudate nucleus, and eight age-matched autopsy controls. RESULTS: The pathology showed three FCD IIa, containing dysmorphic neurons, and one FCD IIb, balloon cells. In the neocortex, the concentrations (each positive cell number/all cell numbers in the evaluated field) of GAD+, CR+ and CB+ cells were significantly lower in FCD than in controls. On the contrary, in the caudate nucleus those of CR+ and CB+ cells were significantly more in FCD than in controls. CONCLUSION: The interneuron imbalance between the neocortex and basal ganglia may affect the epileptogenesis of FCD.

Effects of chronic nicotine on the autocrine regulation of pancreatic cancer cells and pancreatic duct epithelial cells by stimulatory and inhibitory neurotransmitters.

Pancreatic ductal adenocarcinoma (PDAC) has a mortality rate near 100%. Smoking is a documented risk factor. However, the mechanisms of smoking-associated pancreatic carcinogenesis are poorly understood. We have shown that binding of nicotine to nicotinic acetylcholine receptors (nAChRs) expressing subunits α7, α3 and α5 in PDAC and pancreatic duct epithelial cells in vitro triggered the production of the neurotransmitters noradrenaline and adrenaline by these cells. In turn, this autocrine catecholamine loop significantly stimulated cell proliferation via cyclic adenosine 3',5'-monophosphate-dependent signaling downstream of beta-adrenergic receptors. However, the observed responses only represent acute cellular reactions to single doses of nicotine whereas nicotine exposure in smokers is chronic. Using the PDAC cell lines BxPC-3 and Panc-1 and immortalized pancreatic duct epithelial cell line HPDE6-C7, our current experiments reveal a significant sensitization of the nAChR-driven autocrine catecholamine regulatory loop in cells pre-exposed to nicotine for 7 days. The resulting increase in catecholamine production was associated with significant inductions in the phosphorylation of signaling proteins ERK, CREB, Src and AKT, upregulated protein expression of nAChR subunits α3, α4, α5 and α7 and increased responsiveness to nicotine in 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and cell migration assays. All three cell lines produced the inhibitory neurotransmitter γ-aminobutyric acid, an activity inhibited by gene knockdown of the α4ß2nAChR and suppressed by chronic nicotine via receptor desensitization. All of the observed adverse effects of chronic nicotine were reversed by treatment of the cells with γ-aminobutyric acid, suggesting the potential usefulness of this agent for the improvement of PDAC intervention strategies in smokers.

High titre of antiglutamic acid decarboxylase autoantibody is a strong predictor of the development of thyroid autoimmunity in patients with type 1 diabetes and latent autoimmune diabetes in adults.

OBJECTIVE: Type 1 diabetes mellitus (T1DM) is frequently associated with autoimmune thyroid diseases (AITD), but little is known about the risk of AITD in latent autoimmune diabetes in adults (LADA). We evaluated the genetic and immunological factors involved in the development of thyroid autoimmunity in patients with LADA and T1DM. PATIENTS AND MEASUREMENTS: One hundred and ninety T1DM and 135 LADA patients were recruited in the study. Thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TGAb), glutamic acid decarboxylase antibody (GADA) and thyroid function were measured. The cytotoxic-lymphocyte-associated antigen-4 (CTLA-4) +49A/G and CT60 polymorphisms and the human leucocyte antigen (HLA)-DQA1-DQB1 genotype were determined. RESULTS: The prevalence of thyroid antibodies (TGAb and/or TPOAb) and thyroid dysfunction was 27·4% and 9·5% in patients with T1DM, and 21·5% and 11·1% in patients with LADA. Thyroid-antibody-positive T1DM patients had higher frequencies of GADA and HLA-DQA1*03-DQB1*0401 haplotypes than thyroid-antibody negatives (P < 0·05). Thyroid-antibody-positive LADA patients had higher GADA titre, lower C-peptide levels and higher frequencies of HLA-DQA1*03-DQB1*0401 haplotypes (P < 0·05). The CTLA-4 +49A/G and CT60 polymorphism was associated with T1DM complicated with thyroid autoimmunity (OR = 2·33 and 2·54). Logistic regression revealed that only high-titre GADA was associated with development of thyroid autoimmunity in patients with T1DM and LADA (OR = 3·50 and 3·10, respectively), and the presence of thyroid antibody predicted high risk for thyroid dysfunction in patients with T1DM and LADA (OR = 9·25 and 10·70, respectively). CONCLUSION: Regular screening of thyroid antibody and function are recommended, especially in patients with T1DM and LADA with high GADA titre.

Kalirin-7, an important component of excitatory synapses, is regulated by estradiol in hippocampal neurons.

Estradiol enhances the formation of dendritic spines and excitatory synapses in hippocampal neurons in vitro and in vivo, but the underlying mechanisms are not fully understood. Kalirin-7 (Kal7), the major isoform of Kalirin in the adult hippocampus, is a Rho GDP/GTP exchange factor localized to postsynaptic densities. In the hippocampus, both Kal7 and estrogen receptor α (ERα) are highly expressed in a subset of interneurons. Over-expression of Kal7 caused an increase in spine density and size in hippocampal neurons. To determine whether Kalirin might play a role in the effects of estradiol on spine formation, Kal7 expression was examined in the hippocampus of ovariectomized rats. Estradiol replacement increased Kal7 staining in both CA1 pyramidal neurons and interneurons in ovariectomized rats. Estradiol treatment of cultured hippocampal neurons increased Kal7 levels at the postsynaptic side of excitatory synapses and increased the number of excitatory synapses along the dendrites of pyramidal neurons. These increases were mediated via ERα because a selective ERα agonist, but not a selective ERß agonist, caused a similar increase in both Kal7 levels and excitatory synapse number in cultured hippocampal neurons. When Kal7 expression was reduced using a Kal7-specific shRNA, the density of excitatory synapses was reduced and estradiol was no longer able to increase synapse formation. Expression of exogenous Kal7 in hippocampal interneurons resulted in decreased levels of GAD65 staining. Inhibition of GABAergic transmission with bicuculline produced a robust increase in Kal7 expression. These studies suggest Kal7 plays a key role in the mechanisms of estradiol-mediated synaptic plasticity.

Activation of GABAergic neurons following tooth pulp stimulation.

The functional impact of GABA (gamma-aminobutyric acid)ergic neurons in nociceptive transmission of the spinal trigeminal nucleus is not fully established. Using both the glutamic acid decarboxylase (GAD)(67)-green fluorescence protein (GFP) knock-in mouse and the tooth pulp stimulation model, we performed double-immunofluorescent histochemistry to determine the characteristics of GABAergic neuron activation in the spinal trigeminal nucleus. The number of Fos-positive GABAergic neuronal profiles was significantly increased 2 hrs after tooth pulp stimulation. The Fos/GFP double-labeled neurons were mainly present in superficial laminae of the spinal trigeminal subnucleus interpolaris-caudalis transition (Vi/Vc) and subnucleus caudalis (Vc) on the side ipsilateral to the stimulation. Subsequently, the number of double-labeled neurons decreased gradually and became comparable with that of the controls by 48 hrs. Our results provide direct morphological evidence that a subset of GABAergic neurons in the spinal trigeminal system was activated during tooth pulp stimulation.

Presence of human herpes virus 6 DNA exclusively in temporal lobe epilepsy brain tissue of patients with history of encephalitis.

Temporal lobe epilepsy (TLE) is frequently associated with mesial temporal sclerosis (MTS). Many etiologic aspects of TLE are still unresolved. Here, we aimed to analyze the presence of human herpes virus 6 (HHV-6) DNA in distinct TLE pathologies. Nested polymerase chain reaction (PCR) in surgical tissue from 38 pharmaco-resistant TLE patients and 10 autopsy controls revealed HHV-6 DNA in 55.6% of the TLE patients with a history of encephalitis, involving MTS and gliotic hippocampi without substantial neurodegeneration, but not in lesion-associated TLE or nonlesional MTS with or without a history of complex febrile seizures (CFS). HHV-6 protein was present in only one patient's tissue. Our data argue against HHV-6 as a major local pathogenetic factor in MTS hippocampi after CFS. The high detection rate of HHV-6 DNA suggests a potential pathogenetic role of HHV-6 in TLE patients with a history of encephalitis.

Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications.

BACKGROUND: Based on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR) hypothalamic structures in the rat brain, here we investigated: 1.) The cellular and subcellular localization of NTPDase3; 2.) The effects of 17beta-estradiol on the expression level of hypothalamic NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal preparations. METHODS: Combined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode. RESULTS: Combined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD) indicated that gamma-amino-butyric-acid- (GABA) ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial respiration rates with and without the inhibition of NTPDases confirmed the presence of NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and total mitochondrial respiratory capacity. CONCLUSION: Altogether, these results suggest the possibility that NTPDases, among them NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular availability of ATP needed for excitatory neuronal functions including neurotransmission.

Ultrastructural and functional characterization of satellitosis in the human lateral amygdala associated with Ammon's horn sclerosis.

The amygdala displays neuronal cell loss and gliosis in human temporal lobe epilepsy (TLE). Therefore, we investigated a certain type of gliosis, called satellitosis, in the lateral amygdala (LA) of TLE patients with Ammon's horn sclerosis (AHS, n = 15) and non-AHS (n = 12), and in autopsy controls. Satellite cells were quantified using light and electron microscopy at the somata of Nissl-stained and glutamic acid decarboxylase-negative projection neurons, and their functional properties were studied using electrophysiology. Non-AHS cases suffered from ganglioglioma, cortical dysplasia, Sturge-Weber syndrome, astrocytoma WHO III-IV, Rasmussen's encephalitis, cerebral infarction and perinatal brain damage. TLE cases with AHS had a more prominent satellitosis as compared to non-AHS and/or autopsy cases, which correlated with epilepsy duration but not age. At ultrastructural level, the predominant type of satellite cells occurring in both AHS and non-AHS cases displayed a dark cytoplasm and an irregularly shaped dark nucleus, whereas perineuronal glial cells with a light cytoplasm and light oval nucleus were much rarer. Satellite cells expressed time- and voltage-dependent transmembrane currents as revealed by patch-clamp recordings typical for 'complex' glia, although only 44% of satellite cells were immunostained for the chondroitin sulfate proteoglycan NG2. Together, the perineuronal cells described here were a heterogenous cell population regarding their NG2 expression, although they resembled NG2 cells rather than bona fide oligodendrocytes and astrocytes based on their ultrastructural and electrophysiological characteristics. Thus, perineuronal satellitosis as studied in the LA seems to be a hallmark of AHS-associated TLE pathology in patients suffering from intractable epilepsy.

Avaliação da função pancreática em pacientes com diabetes melito tipo 1 de acordo com a duração da doença / Pancreatic function assessment in type 1 diabetes mellitus patients according to disease duration

Os pacientes com diabetes melito tipo 1 (DM1) podem apresentar secreção residual de insulina por longos períodos, o que tem sido associado a prognóstico mais favorável. OBJETIVO: Avaliar a secreção de insulina por meio da dosagem de peptídeo C (PC) em pacientes com DM1 de curta (<5 anos; grupo 1) e longa (> 5 anos; grupo 2) duração da doença. PACIENTES E MÉTODOS: Voluntários com DM1 coletaram sangue em jejum e 6 minutos após a infusão de glucagon para dosagem de PC, HbA1c e anti-GAD. RESULTADOS: Foram avaliados 43 pacientes, 22 no grupo 1 e 21 no grupo 2. Secreção de insulina preservada (PC > 1,5 ng/mL) foi identificada em seis (13,9 por cento) e oito (18,6 por cento) casos nas coletas basal (PC1) e após estímulo (PC2), sem diferença entre os grupos (p = 0,18 e 0,24). PC1 foi detectável (> 0,5 ng/mL) em 13 (30,2 por cento) e PC2 em 18 (41,9 por cento) casos, mais frequentes no grupo 1 do que no 2 (p = 0,045 para PC1/p = 0,001 para PC2). Os títulos de PC1 (1,4 ±0,8 versus 1,2 ±1,0; p = 0,69) ou PC2 (1,8 ±1,5 versus 1,7 ±0,8; p = 0,91) não diferiram entre os grupos. No grupo 1 houve correlação inversa entre tempo de doença e PC2 (R = -0,58; p = 0,025). CONCLUSÃO: Uma proporção significativa dos pacientes com DM1 apresenta secreção residual de insulina, especialmente nos primeiros cinco anos da doença. Tais indivíduos representam a população ideal para estudos visando à prevenção secundária da doença.

Patients with type 1 diabetes (T1D) may exhibit some residual insulin secretion for many years after their diagnosis. This has been associated with a more favorable prognosis. OBJECTIVE: To analyze insulin secretion in individuals with T1D using C-peptide (CP) response to glucagon and comparing patients with recent onset (<5 years - Group 1) and long-standing disease (>5 years -Group 2). METHODS: Subjects with T1D had their blood sampled before (fasting) and 6 minutes after glucagon infusion for CP, HbA1c and anti-GAD measurement. RESULTS: Forty-three individuals were evaluated, 22 in Group 1 and 21 in Group 2. Preserved insulin secretion (CP >1.5 ng/mL) was observed in 6 (13.9 percent) and in 8 (18.6 percent) patients before (CP 1) and after (CP 2) glucagon stimulus, respectively, showing no difference between the groups (p=0.18 and 0.24). CP 1 and CP 2 were detectable (>0.5 ng/dL) in 13 (30.2 percent) and 18 (41.9 percent) patients, respectively. Both were more frequent in Group 1 than in Group 2 (p=0.45 for CP1/p=0.001 for CP 2). Similar serum levels where seen between the groups, both before and after stimulus (1.4±0.8 vs. 1.2±1.0; p=0.69 and 1.8±1.5 vs. 1.7±0.8; p=0.91). Group 1 presented an inverse correlation between disease duration and CP 2 (R=-0.58; p=0.025). CONCLUSION: A significant number of patients with T1D have detectable residual insulin secretion, especially in the first 5 years of disease. These subjects are an ideal population for clinical trials that target the prevention of â cell function loss in T1D.

Diabetes auto-imune latente do adulto ou diabetes melito tipo 2 magro? / Latent autoimmune diabetes of adult or slim type 2 diabetes mellitus?

A prevalência do diabetes auto-imune latente do adulto (LADA) varia em virtude da população estudada, dos critérios usados e dos anticorpos avaliados. Em 256 pacientes com menos de 25 anos, encontramos 26 (10,2 por cento) com anticorpos anti-GAD (GADA) positivos, dos quais 16 (6,3 por cento) evoluíram sem necessidade de insulina inicialmente. Embora exista controvérsias, sugere-se como critérios diagnósticos de LADA: idade entre 25 e 65 anos; ausência de cetoacidose ou hiperglicemia sintomática no diagnóstico ou imediatamente após, sem necessidade de insulina por 6 a 12 meses; e presença de auto-anticorpos (especialmente GADA). A auto-imunidade e a resistência insulínica coexistem no LADA, e a contribuição desses fatores parece estar refletida nos títulos de GADA. Um subgrupo similar aos diabéticos tipo 2, fenotipicamente e na progressão para necessidade de insulina, parece ser melhor identificado pela presença de baixos títulos de GADA, sobretudo isolados. Por outro lado, indivíduos com altos títulos de GADA e múltiplos anticorpos apresentam fenótipo mais próximo do diabetes melito do tipo 1 (DM1) clássico e são de maior risco para falência prematura das células-beta. Comparados aos diabéticos GADA-negativos, pacientes com LADA apresentam maior prevalência de outros auto-anticorpos (anti-TPO, anti-21-hidroxilase e associados à doença celíaca) e maior freqüência de genótipos e haplótipos de risco para DM1. Pacientes com altos títulos de GADA podem ser beneficiados, retardando a falência das células-beta, com a insulinização precoce e evitando-se o uso de sulfoniluréias. Em oposição, pacientes com baixos títulos de GADA aparentemente não teriam prejuízos em serem conduzidos da mesma forma que pacientes portadores de diabetes melito tipo 2 (DM2) (GADA-negativos).

The prevalence of latent autoimmune diabetes of the adult (LADA) varies according to the population studied, criteria used and antibodies analyzed. In a series of 256 patients > 25 years, we found that 26 (10.2 percent) were anti-GAD antibody (GADA) positive and 16 of them (6.3 percent) progressed without initial insulin requirement. Although controversy exists, the following diagnostic criteria for LADA are suggested: age between 25 and 65 years; absence of ketoacidosis or symptomatic hyperglycemia at diagnosis or immediately thereafter, without insulin requirement for 6-12 months; and presence of autoantibodies (especially GADA). Autoimmunity and insulin resistance coexist in LADA and the contribution of these factors seems to be reflected in GADA titers. A subgroup, which is phenotypically and in terms of insulin requirement similar to type 2 diabetic patients, seems to be better identified based on the presence of low GADA titers, especially when these antibodies are present alone. On the other hand, subjects with high GADA titers and multiple antibodies show a phenotype close to that of classical DM 1 and are at a higher risk of premature beta-cell failure. Compared to GADA-negative diabetics, patients with LADA present a higher prevalence of other autoantibodies (anti-TPO, anti-21-hydroxylase and antibodies associated with celiac disease) and a higher frequency of genotypes and haplotypes indicating a risk for DM 1. Patients with high GADA titers may benefit from early insulinization and avoiding the use of sulfonylureas, delaying beta-cell failure. In contrast, patients with low GADA titers do not seem to have any disadvantage when managed as type 2 diabetic patients (GADA negative).

The importance of CTLA-4 polymorphism and human leukocyte antigen genotype for the induction of diabetes-associated cytokine response in healthy school children.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease associated with the destruction of pancreatic beta cells and genetically linked to human leukocyte antigen (HLA) class II DR3-DQ2 and DR4-DQ8 haplotypes. The +49A/G polymorphism of the immunoregulatory cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene is also associated with T1D. Genetic and environmental risk factors precede the onset of T1D, which is characterized by a T helper 1 cell-dominating cytokine response to diabetes-related autoantigens. AIM: To investigate immunological differences between healthy children with and without CTLA-4 +49A/G and HLA genetic susceptibility for T1D. STUDY DESIGN: Young, 7-15 years of age, healthy subjects (n = 58) were investigated to test whether CTLA-4 +49A/G genotype was associated with enzyme-linked immunospot assay T-cell responses to T1D-related autoantigens. Because T1D is primarily HLA-DQ associated, we stratified the healthy subjects by HLA genotypes associated with the disease. RESULTS: Peptide of heat shock protein 60 induced a higher interferon-gamma (IFN-gamma) response in subjects with risk-associated CTLA-4 polymorphism (GG genotype) (p = 0.02) while glutamic acid decarboxylase 65-induced interleukin-4 (IL-4) secretion was lower in GG genotype subjects (p = 0.02). CONCLUSION: The increased IFN-gamma response and lower IL-4 response toward diabetes-related autoantigens shown in CTLA-4 +49 GG risk subjects show a possible mechanism for the association between CTLA-4 and T1D.

In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism.

BACKGROUND AND PURPOSE: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503. EXPERIMENTAL APPROACH: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907. KEY RESULTS: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity. CONCLUSIONS AND IMPLICATIONS: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.

Preoptic area neurons and the homeostatic regulation of rapid eye movement sleep.

The median preoptic nucleus (MnPN) and the ventral lateral preoptic area (vlPOA) of the hypothalamus express sleep-related Fos immunoreactivity, and a subset of Fos-immunoreactive neurons (IRNs) in these nuclei contain glutamic acid decarboxylase (GAD), a marker of GABAergic cells. We recently showed that the numbers of Fos-positive (Fos+) and Fos+ GAD-IRNs in both the MnPN and the vlPOA are positively correlated with the total amount of preceding sleep. The present study was designed to clarify whether or not activation of sleep-related neurons in the rat MnPN and vlPOA is associated with rapid eye movement (REM) sleep regulation. Expression of c-fos in MnPN and vlPOA neurons was examined under conditions of spontaneous sleep, REM sleep restriction, and REM sleep recovery after REM sleep restriction. Across all conditions, the number of Fos-IRNs was highest in REM-sleep-restricted rats displaying the highest levels of REM sleep homeostatic pressure/drive, i.e., those rats exhibiting the most frequent attempts to enter REM sleep. This finding provides the first evidence that activation of subsets of MnPN and vlPOA neurons is more strongly related to REM sleep pressure than to REM sleep amount.

New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors.

Ongoing neurogenesis in the adult mammalian dentate gyrus and olfactory bulb is generally accepted, but its existence in other adult brain regions is highly controversial. We labeled newly born cells in adult rats with the S-phase marker bromodeoxyuridine (BrdU) and used neuronal markers to characterize new cells at different time points after cell division. In the neocortex and striatum, we found BrdU-labeled cells that expressed each of the eight neuronal markers. Their size as well as staining for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase 67, calretinin and/or calbindin, suggest that new neurons in both regions are GABAergic interneurons. BrdU and doublecortin-immunoreactive (BrdU+/DCX+) cells were seen within the striatum, suggesting migration of immature neurons from the subventricular zone. Surprisingly, no DCX+ cells were found within the neocortex. NG2 immunoreactivity in some new neocortical neurons suggested that they may instead be generated from the NG2+ precursors that reside within the cortex itself.