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SummaryBackgroundAn increasing burden of invasive group A streptococcal infections is reported in multiple countries, notably England, where scarlet fever cases are also abundant. In England, increased scarlet fever and invasive infections have been associated with emergence of a sublineage ofemm1Streptococcus pyogenesthat expresses increased SpeA scarlet fever erythrogenic toxin. Wider surveillance for toxigenicStreptococcus pyogeneslineage M1UKis much needed however, to date, lineage assignment has required genome sequencing limiting surveillance to those centres with access to such facilities.MethodsTo circumvent the requirement for genome sequencing, an allele-specific PCR was developed to distinguish M1UKfrom otheremm1 strains. Additional PCR assays were developed to distinguish M1UKfrom two intermediate lineages that were detected previously. The assay was evaluated using DNA from genome-sequenced upper respiratory tractemm1S. pyogenesstrains and a further set of 16 genome-sequenced invasiveS. pyogenesisolates that included the two intermediate lineages. The assay was then applied to DNA from all 305 invasiveemm1 isolates that had been submitted to the reference laboratory in the one pear period Jan 1-Dec 31 2020, in order to assign lineage.ResultsThe allele specific PCR was 100% accurate when compared with genome sequencing, correctly identifying M1UK, two intermediate sublineages, and otheremm1 strains. The assay demonstrated the M1UKlineage to be dominant amongemm1 invasive isolates in England, representing 278/305 (91%) of invasiveemm1 isolates by end of 2020.ImplicationsEmm1S. pyogeneshave a prominent role in invasive infections; anyemm1 lineage that demonstrates enhanced fitness within the population is of public health concern. The allele specific PCR provides a readily available method to subtypeemm1 isolates and does not require access to complex sequencing facilities. The data confirm that the M1UKlineage has persisted and further expanded in England underlining the importance of wider global surveillance.

The present study was designed to investigate the effects of long-term exposure (4 weeks) to the widely used narcotic drug and putative neurotoxicant 3,4-methylenedioxymetamphetamine (MDMA; “ecstasy”) on neuronal transmitter transport and progression of experimental periodontitis in male Wistar rats. The rats were exposed to MDMA (10 mg/kg/day i.p.) or saline five days a week for four consecutive weeks. Exposure to MDMA induced a significant reduction in the synaptosomal reuptake of serotonin, while the uptake of dopamine was significantly increased 24 h after the last injection of MDMA. In contrast, the synaptosomal uptake of noradrenaline and the vesicular uptake through the vesicular monoamine transporter 2 were not affected. In the experiments of periodontitis development, ligature-induced periodontitis was induced three days prior to MDMA administration. Compared to controls, MDMA-treated rats developed significantly more periodontitis. In conclusion, our results show that long-term exposure to MDMA affects the serotonergic and dopaminergic transport systems in the rat brain and increased the susceptibility to the psychosomatic ailment periodontitis following disturbances of brain immune-regulatory systems. These results are interesting with respect to recent research showing that changes in neurotransmitter signalling may alter the reactivity of brain-controlled immunoregulatory systems controlling pathogenic microorganisms colonizing mucosal surfaces.

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and 13C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. 13C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.

Current treatment of nerve agent poisoning consists of prophylactic administration of pyridostigmine and therapy using atropine, an oxime and a benzodiazepine. Pyridostigmine does however not readily penetrate the blood-brain barrier giving ineffective protection of the brain against centrally mediated seizure activity. In this study, we have evaluated donepezil hydrochloride, a partial reversible inhibitor of acetylcholinesterase (AChE) clinically used for treating Alzheimer's disease, in combination with procyclidine, used in treatment of Parkinson's disease and schizophrenia, as prophylaxis against intoxication by the nerve agent soman. The results demonstrated significant protective efficacy of donepezil (2.5 mg/kg) combined with procyclidine (3 or 6 mg/kg) when given prophylactically against a lethal dose of soman (1.6 x LD(50)) in Wistar rats. No neuropathological changes were found in rats treated with this combination 48 h after soman intoxication. Six hours after soman exposure cerebral AChE activity and acetylcholine (ACh) concentration was 5% and 188% of control, respectively. The ACh concentration had returned to basal levels 24 h after soman intoxication, while AChE activity had recovered to 20% of control. Loss of functioning muscarinic ACh receptors (17%) but not nicotinic receptors was evident at this time point. The recovery in brain AChE activity seen in our study may be due to the reversible binding of donepezil to the enzyme. Donepezil is well tolerated in humans, and a combination of donepezil and procyclidine may prove useful as an alternative to the currently used prophylaxis against nerve agent intoxication.

The purpose of this study was to use an established porcine model to investigate the effects on immune function of severe gunshot injury.Twelve pigs sustained two standardised rounds, one through right femur and one through left upper abdomen. First aid treatment and acute surgery was started immediately. Blood samples were drawn before shooting and after 75 min. Circulating neutrophils were isolated and reactive oxygen species (ROS) measured. Serum levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and IL-10 were determined at 0, 75 min, as well as 2h after incubation with 1 microg/ml endotoxin in an ex vivo whole blood model.TNF-alpha, IL-1beta, and IL-6 significantly increased at 75 min. ROS in circulating granulocytes tended to increase (NS). Incubation with endotoxin led to a more than 100-fold increase of TNF-alpha pre-trauma, compared to a three-fold increase post-trauma (p0.0001 between groups). A similar pattern was obtained for IL-1beta, and IL-6. IL-10 was below detection in all samples. The granulocytes maintained their ability to react to the protein kinase C activator phorbol myristate acetate (PMA) after trauma.Severe gunshot injury and peritraumatic stress rapidly activate circulating immune cells, but reduce their capacity to react to a subsequent challenge to endotoxin.

Factor XI (FXI) deficiency is associated with an abnormal bleeding state. The extent of bleeding does not correlate well with the plasma concentration of FXI, and it has been suggested that also unknown factors interfere with the bleeding tendency. In a recent paper ( Thromb. Res. 74, 477–485, 1994) we found that FXIa activated in human plasma was present in association with part of factor XIIa (FXIIa) and part of kallikrein, influencing their functional activities. Should the activity level of FXIa also be altered by the other contact factors this might provide one approach to the problem of the failure of assays of FXIa to correlate with bleeding tendency. In the present study we have developed an assay procedure for FXIa based on its amidolytic (S-2366) activity, and allowing at the same time a quantification of the amount of FXIa associated to kallikrein. The total amidase activity obtained was separated into two main fractions by use of soybean trypsin inhibtor (STI), corn inhibitor (CI) and lima bean trypsin inhibitor (LTI). One fraction contained free FXIa which could be specifically blocked by LTI. An inhibitor resistant fraction was found to contain FXIa inactive in association with kallikrein. The content of FXIa could be assessed in experiments with mixtures of normal plasma and plasma deficient in prekallikrein, and was taken into account in the calculations. This fraction increased during storage of plasma at −70°C. To obtain stable and comparable assay conditions the method was based on plasma stored for at least four weeks. The specificity of the method was verified by parallel radial immunodiffusion tests. The results imply that the activity level of FXIa is dependent on kallikrein present. If the experimental results has relevance to the situation under physiological conditions, they indicate one possible cause of the failure of assays of FXI to correlate with bleeding tendency.

The aim of this study was to examine the importance of the vesicle-associated synapsin I and II phosphoproteins for the accumulation of neurotransmitters in central cholinergic as compared to central glutamatergic and GABAergic nerve terminals. In brain homogenate samples from mice devoid of synapsin I and II, the levels of vesicular transporters for glutamate (VGLUT1-2) and GABA (VGAT) were decreased by 35-40% in striatum and cortex, while no change was apparent for the vesicular acetylcholine transporter (VAChT). The severe decrease in the levels of amino acid vesicular transporters caused only minor changes in the concentrations of the respective neurotransmitters in homogenates of the three selected brain areas from synapsin I- and II-deficient mice. However, when measured in a crude vesicular fraction, the concentrations of glutamate and GABA were decreased by 48-60% in synapsin-deficient mice, with a similar decrease in the levels of VGLUT1, VGLUT2 and VGAT. In comparison, the concentration of acetylcholine and the level of VAChT were not significantly different from wild-type in the vesicular fraction. No changes were seen in the activity of specific enzymes involved in the synthesis of acetylcholine, glutamate or GABA, however, immunoblotting indicated a decrease in the protein level of glutamic acid decarboxylase, isoform 65 (GAD(65)). In conclusion, the results indicate that neurotransmitter regulation in central cholinergic synapses may be less dependent on synapsin I and II compared to the marked alterations seen in the glutamatergic and GABAergic synapses.

The cerebellar granule cells have been extensively used for studies on metabolism, neurotransmission and neurotoxicology, since they can easily be grown in cultures. However, knowledge about the development of different proteins essential for synaptic transmission in these cells is lacking. This study has characterized the developmental profiles of the vesicular glutamate transporters (VGLUTs) and the synaptic vesicle proteins synapsins and synaptophysin in cerebellar granule cells and in co-cultures containing both granule cells and astrocytes. The protein levels of VGLUT2 decreased by approximately 70% from days 2 to 7 in vitro, whereas the levels of VGLUT1 increased by approximately 95%. Protein levels of synapsin I, synapsin IIIa and synaptophysin showed a developmental pattern similar to VGLUT1 while synapsin II and VGLUT3 were absent. The mRNA expressions of VGLUT1 and VGLUT2 were in accordance with the protein levels. The results indicate both that cerebellar granule cells are mature at approximately 7 days in vitro, and that the up-regulation of VGLUT1 and down-regulation of VGLUT2 in cerebellar granule cells are both independent of surrounding astrocytes and neuronal input. The results of this study are discussed in relation to general developmental profiles of VGLUTs in other brain regions.

Donepezil is a selective inhibitor of acetylcholinesterase (AChE) clinically used for treating Alzheimer’s disease. Cholinergic effects after short-term exposure of donepezil (up to 12 h) have been extensively studied in rats, but few have addressed the potential long-term effects. After 14 days administration (1×3 mg/kg, decapitation 4 h after the last injection) the cerebral acetylcholine level was increased by 35% and the AChE activity was decreased by 66% and 32% in brain and blood, respectively. No change was detected in choline acetyltransferase activity, or the levels of vesicular acetylcholine transporter, choline transporter, or muscarinic receptors. Expression of various cholinergic genes was unaffected. Preliminary results of AChE activity in human blood showed 60–97% and 43–89% of pre-exposed level after one and three days of donepezil administration (5 mg daily), respectively. In conclusion, donepezil exposure in rats at doses that do not inhibit brain AChE continuously during the day, will not lead to tolerance development.

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a commonly abused drug which has been shown to be neurotoxic to serotonergic neurons in many species. The exact mechanism responsible for the neurotoxicity of MDMA is, however, poorly understood. In this study, the effects of MDMA on the synaptosomal and vesicular uptake of neurotransmitters were investigated. Our results show that MDMA (0.5-20 microM) reduces both synaptosomal and vesicular uptake of serotonin and dopamine in a dose dependent manner in vitro, while the uptake of glutamate and gamma-aminobutyric acid (GABA) remains unaffected. Ex vivo experiments support the importance of the monoamines, with predominant dopaminergic inhibition at short-term exposure (3 x 15 mg/kg; 2-h intervals), and exclusively serotonergic inhibition at long-term exposure (2 x 10 mg/kg per day; 4 days). This study also compares MDMA and the structurally related antidepressant paroxetine, in an attempt to reveal possible cellular mechanisms for the serotonergic toxicity of MDMA. One important difference between paroxetine and MDMA is that only MDMA has the capability of inhibiting vesicular uptake of monoamines at doses used. We suggest that inhibition of the vesicular monoamine transporter-2, and a following increase in cytoplasmatic monoamine concentrations, might be crucial for the neurotoxic effect of MDMA.

Denne hovedoppgaven omhandler ecstasys effekter på neurotransmitterbalansen i hjernen hos rotte. Ecstasy (3,4-metylendioksymetamfetamin; MDMA) er et amfetaminliknende syntetisk stoff og bruken av stoffet blant ungdommer har økt betydelig de senere årene. MDMA virker både oppkvikkende (som amfetamin) og hallusinerende (som LSD) og bruken er blitt nært tilknyttet house- og rave-kulturen. MDMA er vist å påvirke neurotransmitterbalansen i hjernen ved å stimulere frigjøring og hemme reopptak av serotonin (5-HT) og dopamin (DA) fra den synaptiske spalten. MDMAs effekt på synaptosomalt og vesikulært neurotransmitteropptak ble testet både in vitro og in vivo. In vitro administrasjon av MDMA viste betydelig nedsatt reopptak av 5-HT og DA i synaptosomer og synaptiske vesikler. Korttidsforsøk in vivo forårsaket reduksjon av DA-opptak i synaptosomer, mens opptak av 5-HT til sammenligning ikke ble signifikant påvirket. Langtidsforsøk in vivo viste betydelig nedsatt opptak av 5-HT i synaptosomer og vesikler, mens DA ikke ble påvirket i signifikant grad.Både in vitro og in vivo-forsøkene viser at MDMA påvirker opptak av neurotransmitterne DA og 5-HT på en negativ måte. Utfra egne og andres resultater er det holdepunkter for å anta at MDMA kan skape både midlertidige og permanente skader i hjernen. Som en sammenligning med MDMAs effekter på neurotransmitteropptak ble det utført in vitro- og in vivo-forsøk med den selektive serotonin reopptakshemmeren paroxetin. Paroxetin hemmet signifikant både synaptosomalt og vesikulært opptak av 5-HT og DA etter in vitro-eksponering, men hadde ingen effekt på opptak etter in vivo-administrasjon. Det ble også utført in vitro-forsøk med eksponering for metamfetamin og perfluorooktansulfonat for sammenligning med in vitro-forsøk med MDMA.