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Microglial cells constantly surveil the cerebral microenvironment and become activated following injury and disease to mediate inflammatory responses. The nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome, which is abundantly expressed in microglial cells, plays a key role in these responses as well as in the development of many neurological disorders. Microglial cell lines are a valuable tool to study the causes and possible treatments for neurological diseases which are linked to inflammation. Here, we investigated whether the mouse microglial cell line IMG is suitable to study NLRP3 inflammasome by incubating cells with different concentrations of NLRP3 inflammasome priming and activating agents lipopolysaccharide (LPS) and ATP, respectively, and applying short (4 h) or long (24 h) LPS incubation times. After short LPS incubation, the mRNA levels of most pro-inflammatory and NLRP3 inflammasome-associated genes were more upregulated than after long incubation. Moreover, the combination of higher LPS and ATP concentrations with short incubation time resulted in greater levels of active forms of caspase-1 and interleukin-1 beta (IL-1β) proteins than low LPS and ATP concentrations or long incubation time. We also demonstrated that treatment with NLRP3 inflammasome inhibitor glibenclamide suppressed NLRP3 inflammasome activation in IMG cells, as illustrated by the downregulation of gasdermin D N-fragment and mature caspase-1 and IL-1β protein levels. In addition, we conducted similar experiments with primary microglial cells and BV-2 cell line to determine the similarities and differences in their responses. Overall, our results indicate that IMG cell line could be a valuable tool for NLRP3 inflammasome studies. In IMG cells, 4-h incubation with lipopolysaccharide (LPS) induces a stronger upregulation of NLRP3 inflammasome-associated pro-inflammatory genes compared to 24-h incubation. NLRP3 inflammasome is robustly activated only after the addition of 3 mM of ATP following short LPS incubation time., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Several reports have provided crucial evidence in animal models that epigenetic modifications, such as DNA methylation, may be involved in psychostimulant-induced stable changes at the cellular level in the brain. Epigenetic editors DNA methyltransferases (DNMTs) and ten-eleven translocation enzymes (TETs) coordinate expression of gene networks, which then manifest as long-term behavioural changes. However, the extent to which aberrant DNA methylation is involved in the mechanisms of substance use disorder in humans is unclear. We previously demonstrated that cocaine modifies gene transcription, via DNA methylation, throughout the brain and in peripheral blood cells in mice., Results: We treated human peripheral blood mononuclear cells (PBMCs) from healthy male donors (n = 18) in vitro with psychostimulants (amphetamine, cocaine). After treatment, we assessed mRNA levels and enzymatic activities of TETs and DNMTs, conducted genome-wide DNA methylation assays and next-generation sequencing. We found that repeated exposure to psychostimulants decreased mRNA levels and enzymatic activity of TETs and 5-hydroxymethylation levels in PBMCs. These data were in line with observed hyper- and hypomethylation and mRNA expression of marker genes (IL-10, ATP2B4). Additionally, we evaluated whether the effects of cocaine on epigenetic editors (DNMTs and TETs) and cytokines interleukin-6 (IL-6) and IL-10 could be reversed by the DNMT inhibitor decitabine. Indeed, decitabine eliminated cocaine's effect on the activity of TETs and DNMTs and decreased cytokine levels, whereas cocaine increased IL-6 and decreased IL-10., Conclusions: Our data suggest that repeated psychostimulant exposure decreases TETs' enzymatic activity in PBMCs. Co-treatment with decitabine reversed TETs' levels and modulated immune response after repeated cocaine exposure. Further investigation is needed to clarify if TET could represent a putative biomarker of psychostimulant use and if DNMT inhibition could have therapeutic potential., (© 2022. The Author(s).)

Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased Dnmt1 , -3a , and -3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system ( Pdyn, Kor ) and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Niinep, Anier, Eteläinen, Piepponen and Kalda.)

Chronic-pain patients often suffer from depression. In rodent models of neuropathic pain, animals develop depression-like and anxiety behaviors, indicating a relationship between chronic pain and affective disorders. However, the underlying neurobiological mechanisms linking chronic pain and depression are not yet fully understood. Neurogenesis in the hippocampus is a fundamental process related to brain plasticity. Reduced neurogenesis has been associated with the development of mood disorders and cognitive impairments. The current study aims to elucidate the underlying long-term changes in brain plasticity induced by neuropathic pain in mice at a time point when depression-like behavior has already developed. Furthermore, our focus is set on alterations in neurogenesis in the hippocampus. We found that manifestation of anxiety- and depressive-like behavior as well as cognitive impairment co-occur with decreased survival of newly generated cells but not with impaired proliferative activity or reduced number of immature neurons in the dentate gyrus area of the hippocampus. Moreover, we detected an impairment of differentiation of newly generated cells into mature calbindin-positive neurons, accompanied with a shift towards increased differentiation into astroglial cells. These findings indicate that a reduction in mature functional neurons, rather than reduced proliferation or neuronal progenitor cells, are the long-term changes in hippocampal plasticity that manifest in neuropathic pain conditions after depression-like behavior has developed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Individual differences in behavioural traits influence susceptibility to addictive disorders. Drug addiction involves changes in gene expression, proposed to occur via DNA methylation (DNAm)., Aims: To investigate DNAm changes in reward-related brain structures (nucleus accumbens (NAc), lateral habenula (LHb)) in response to cocaine exposure in rats differing in spontaneous exploratory activity., Methods: Rats were observed in the exploration box and categorised as high- (HE) or low explorers (LE). Rats were administered vehicle or cocaine (12 mg/kg, i.p.) for 7 days, followed by a 14-day withdrawal period and cocaine challenge (7 mg/kg); horizontal locomotor activity was recorded. Brain tissue was dissected after 24 h; we analysed messenger RNA (mRNA) and activity levels of epigenetic DNA modifiers (DNMTs and TETs) as well as mRNA and promoter methylation levels at selected genes previously linked to addictive behaviours., Results: The cocaine challenge dose stimulated locomotor activity in both LE- and HE rats only when administered after a repeated cocaine schedule, suggesting development of behavioural sensitisation. Quantitative polymerase chain reaction analyses demonstrated higher basal expression of Dnmt3a, Tet2 and Tet3 in the LHb of HE- vs. LE rats, and we observed differential effects of cocaine exposure on the expression and activity of epigenetic DNA modifiers in the NAc and LHb of HE- and LE rats. Furthermore, cocaine exposure differentially altered promoter methylation levels of A 2A R, Ppp1cc , and Taar7b in the NAc and LHb of HE- and LE rats., Conclusions: DNAm might play a role in the HE- and LE phenotypes as well as mediate behavioural effects of LE- and HE rats in response to drugs of abuse.

An increasing number of reports have provided crucial evidence that epigenetic modifications, such as DNA methylation, may be involved in initiating and establishing psychostimulant-induced stable changes at the cellular level by coordinating the expression of gene networks, which then manifests as long-term behavioral changes. In this study, we evaluated the enzyme activity of DNA methyltransferases (DNMTs) after cocaine treatment and during withdrawal. Furthermore, we studied how genetic or pharmacological inhibition of DNMTs in mouse nucleus accumbens (NAc) affects the induction and expression of cocaine-induced behavioral sensitization. Our results showed that after silencing Dnmt3a in the NAc during the induction phase of cocaine-induced sensitization, overall DNMT activity decreases, correlating negatively with behavioral sensitization. Reduced Dnmt3a mRNA during this phase was the largest contributing factor for decreased DNMT activity. Cocaine withdrawal and a challenge dose increased DNMT activity in the NAc, which was associated with the expression of behavioral sensitization. Long-term selective Dnmt3a transcription silencing in the NAc did not alter DNMT activity or the expression of cocaine-induced behavioral sensitization. However, bilateral intra-NAc injection of a non-specific inhibitor of DNMT (RG108) during withdrawal from cocaine decreased DNMT activity in the NAc and had a small effect on the expression of cocaine-induced behavioral sensitization. Thus, cocaine treatment and withdrawal is associated with biphasic changes in DNMT activity in the NAc, and the expression of behavioral sensitization decreases with non-selective inhibition of DNMT but not with selective silencing of Dnmt3a., (© 2019 Society for the Study of Addiction.)

Early life stress initiates long-term neurobiological changes that affect stress resilience and increased susceptibility to psychopathology. Maternal separation (MS) is used to cause early life stress and it induces profound neurochemical and behavioral changes that last until adulthood. The molecular pathways of how MS affects the regulation of DNA methyltransferases (Dnmt) in brain have not been entirely characterized. We evaluated MS effects on Dnmt1, Dnmt3a and Dnmt3b expression, DNMT enzyme activity and glucocorticoid receptor (GR) recruitment to different Dnmt loci in the prefrontal cortex (PFC) of Wistar rats. We found increased plasma corticosterone levels after MS that were associated with induced Dnmt expression and enzyme activity in rat PFC at post-natal day 15 (PND15). Chromatin immunoprecipitation showed increased binding of GR at the Dnmt3b promoter after MS, suggesting that genomic signaling of GR is an important regulatory mechanism for the induced Dnmt3b expression and DNMT activity. Although GR also binds to Dnmt3a promoter and a putative regulatory region in intron 3 in rat PFC, its expression after maternal separation may be influenced by other mechanisms. Therefore, GR could be a link between early life stress experience and long-term gene expression changes induced by aberrant DNA methylation.

Cocaine-related DNA methylation studies have primarily focused on the specific brain regions associated with drug addiction (e.g., the nucleus accumbens, NAc). To date, no studies have focused on the complex role of both DNA methylation and demethylation in the mechanisms of psychostimulant-induced addiction in the brain and peripheral tissues. Therefore, in this study, we evaluated cocaine treatment and withdrawal (animals were withdrawn from seven days of repeated injections of cocaine that caused behavioral sensitization) effects on epigenetic DNA modifiers (i.e., DNA methyltransferases, [DNMTs] and ten-eleven translocation enzymes [TETs]) in an addiction-specific brain region (NAc), a structure outside the mesolimbic dopaminergic system (cerebellum, Cer), and in peripheral blood cells (PBCs). Using a mouse behavioral sensitization model, we demonstrated that acute cocaine (AC; 0.5 h) treatment significantly decreased Dnmt1, Dnmt3a, Tet1, and Tet2 mRNA levels in the NAc and PBC, whereas at 24 h after AC treatment, Dnmt mRNA expression and enzyme activity levels were significantly increased. Acute procaine treatment caused the opposite effect on the Dnmt3a mRNA level in PBCs; this outcome suggests that the inhibition of voltage-gated sodium channels may be the mechanism that alters Dnmt expression in PBCs. Cocaine withdrawal is associated with increased expression of Dnmts in the NAc, Cer and PBCs and the decreased expression of Tet1 and Tet3 in the NAc. Additionally, cocaine withdrawal increased DNMT but decreased TET activity levels, and these changes were associated with enhanced global and selected candidate gene promoter-region DNA methylation and hydroxymethylation levels in the NAc and PBCs. Together, these data indicate that cocaine treatment and withdrawal affect the expression of epigenetic DNA modifiers in both addiction-specific brain structures and structures outside of the mesolimbic dopaminergic system and PBCs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Membrane-associated glycoprotein neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) play an important role in brain plasticity by regulating cell-cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y neuroblastoma cell line with stable overexpression of PREP, we found a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and NCAM140 protein species, and a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa in PREP-overexpressing cells. Moreover, increased levels of NCAM fragments were found in the concentrated medium derived from PREP-overexpressing cells. PREP overexpression selectively induced an activation of matrix metalloproteinase-9 (MMP-9), which could be involved in the observed degradation of NCAM, as MMP-9 neutralization reduced the levels of NCAM fragments in cell culture medium. We propose that increased PREP levels promote epidermal growth factor receptor (EGFR) signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellular plasticity through NCAM and PSA-NCAM., (© 2016. Published by The Company of Biologists Ltd.)

Mood disorders are associated with alterations in serotonergic system, deficient BDNF (brain-derived neurotrophic factor) signaling and abnormal synaptic plasticity. Increased degradation and reduced functions of NCAM (neural cell adhesion molecule) have recently been associated with depression and NCAM deficient mice show depression-related behavior and impaired learning. The aim of the present study was to investigate potential changes in serotonergic and BDNF systems in NCAM knock-out mice. Serotonergic nerve fiber density and SERT (serotonin transporter) protein levels were robustly reduced in the hippocampus, prefrontal cortex and basolateral amygdala of adult NCAM(-)(/-) mice. This SERT reduction was already evident during early postnatal development. [(3)H]MADAM binding experiments further demonstrated reduced availability of SERT in cell membranes of NCAM(-)(/-) mice. Moreover, the levels of serotonin and its major metabolite 5-HIAA were down regulated in the brains of NCAM(-)(/-) mice. NCAM(-)(/-) mice also showed a dramatic reduction in the BDNF protein levels in the hippocampus and prefrontal cortex. This BDNF deficiency was associated with reduced phosphorylation of its receptor TrkB. Importantly, chronic administration of antidepressant amitriptyline partially or completely restored these changes in serotonergic and BDNF systems, respectively. In conclusion, NCAM deficiency lead to prominent and persistent abnormalities in brain serotonergic and BDNF systems, which likely contributes to the behavioral and neurobiological phenotype of NCAM(-/-) mice., (Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.)

Early life stress is known to promote long-term neurobiological changes, which may underlie the increased risk of psychopathology. Maternal separation (MS) is used as an early life stressor that causes profound neurochemical and behavioural changes in the pups that persist into adulthood. However, the exact mechanism of how MS alters these behavioural changes is not yet understood. Epigenetic modifications, such as DNA methylation, are critical regulators of persistent gene expression changes and may be related to behavioural disorders. The aim of the present study was to investigate whether early life stress on rats could alter cocaine-induced behavioural sensitisation in adulthood via aberrant DNA methylation. We have three main findings: (1) MS increased DNA methyltransferases (DNMTs) expression in the nucleus accumbens (NAc) of infant and adult rats; (2) MS induced DNA hypomethylation on a global level in the NAc, and hypermethylation of the promoter regions of the protein phosphatase 1 catalytic subunit (PP1C) and adenosine A2Areceptor (A2AR) genes, which was associated with their transcriptional downregulation in the NAc; (3) MS-induced molecular changes paralleled an increased response to cocaine-induced locomotor activity and exploratory behaviour in adult rats. Thus, our results suggest that stressful experiences in early life may create a background, via aberrant DNA methylation, which promotes the development of cocaine-induced behavioural sensitisation in adulthood., (© 2013 Elsevier B.V. and ECNP. All rights reserved.)

Several studies suggest that individual variability is a critical component underlying drug addiction as not all members of a population who use addictive substance become addicted. There is evidence that the overall epigenetic status of a cell (epigenome) can be modulated by a variety of environmental factors, such as nutrients and chemicals. Based on these data, our aim was to investigate whether environmental factors like S-adenosylmethionine (SAM) via affecting epigenome could alter cocaine-induced gene expression and locomotor sensitization in mice. Our results demonstrate that repeated SAM (10 mm/kg) pretreatment significantly potentiated cocaine-induced locomotor sensitization. Using mouse nucleus accumbens (NAc) tissue, whole-genome gene expression profiling revealed that repeated SAM treatment affected a limited number of genes, but significantly modified cocaine-induced gene expression by blunting non-specifically the cocaine response. At the gene level, we discovered that SAM modulated cocaine-induced DNA methylation by inhibiting both promoter-associated CpG-island hyper- and hypomethylation in the NAc but not in the reference tissue cerebellum. Finally, our in vitro and in vivo data show that the modulating effect of SAM is in part due to decreased methyltransferase activity via down-regulation of Dnmt3a mRNA. Taken together, our results suggest that environmental factors that affect the NAc-cell epigenome may alter the development of psychostimulant-induced addiction and this may explain, at least partly, why some individuals are more vulnerable to drug addiction.

Systemic or intracerebral administration of kainic acid in rodents induces neuronal death followed by a cascade of neuroplastic changes in the hippocampus. Kainic acid-induced neuroplasticity is evidenced by alterations in hippocampal neurogenesis, dispersion of the granule cell layer and re-organisation of mossy fibres. Similar abnormalities are observed in patients with temporal lobe epilepsy and, therefore, kainic acid-induced hippocampal neuroplasticity might mimic pathological mechanisms leading to the formation of 'epileptic brain' in patients with temporal lobe epilepsy. Previous studies have demonstrated that selective serotonin re-uptake inhibitor antidepressants might reduce the severity of seizures in epileptic patients and reduce neuronal death in laboratory animal models of kainic acid-induced neurotoxicity. In the present study, we investigated whether kainic acid-induced neuroplasticity in mice is modulated by the repeated administration of citalopram, a selective serotonin reuptake inhibitor. We found that at the histopathological level, repeated citalopram treatment counteracted the kainic acid-induced neuronal loss and dispersion of young granule neurons expressing the polysialylated neural cell adhesion molecule within the granule cell layer of the hippocampus. Citalopram also counteracted the downregulation of reelin on both mRNA and protein levels induced by kainic acid administration. Our findings indicate that repeated administration of citalopram is able to prevent kainic acid-induced abnormal brain plasticity and thereby prevent the formation of an epileptic phenotype., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The behavioral sensitization produced by repeated cocaine treatment represents the neural adaptations underlying some of the features of addiction in humans. Cocaine administrations induce neural adaptations through regulation of gene expression. Several studies suggest that epigenetic modifications, including DNA methylation, are the critical regulators of gene expression in the adult central nervous system. DNA methylation is catalyzed by DNA methyltransferases (DNMTs) and consequent promoter region hypermethylation is associated with transcriptional silencing. In this study a potential role for DNA methylation in a cocaine-induced behavioral sensitization model in mice was explored. We report that acute cocaine treatment caused an upregulation of DNMT3A and DNMT3B gene expression in the nucleus accumbens (NAc). Using methylated DNA immunoprecipitation, DNA bisulfite modification, and chromatin immunoprecipitation assays, we observed that cocaine treatment resulted in DNA hypermethylation and increased binding of methyl CpG binding protein 2 (MeCP2) at the protein phosphatase-1 catalytic subunit (PP1c) promoter. These changes are associated with transcriptional downregulation of PP1c in NAc. In contrast, acute and repeated cocaine administrations induced hypomethylation and decreased binding of MeCP2 at the fosB promoter, and these are associated with transcriptional upregulation of fosB in NAc. We also found that pharmacological inhibition of DNMT by zebularine treatment decreased cocaine-induced DNA hypermethylation at the PP1c promoter and attenuated PP1c mRNA downregulation in NAc. Finally, zebularine and cocaine co-treatment delayed the development of cocaine-induced behavioral sensitization. Together, these results suggest that dynamic changes of DNA methylation may be an important gene regulation mechanism underlying cocaine-induced behavioral sensitization.

Background: Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme involved in the metabolism of thiopurine drugs. To date, at least 25 single nucleotide polymorphisms have been reported in the TPMT gene, 23 of these are associated with reduced enzyme activity., Methods: The aim of the present study was to sequence the whole coding region of TPMT (exons 3-10) to identify known and novel TPMT sequence variants amongst healthy Estonians. Erythrocyte TPMT activity was also measured to carry out a genotype-phenotype comparison., Results: A total of 21 subjects were heterozygous for known TPMT alleles (*2, *3A, *3C, *9, *12). Several other previously described intronic and exon polymorphisms were identified. Three novel mutations were detected -30T>A in exon 3, 10A>G in intron 3, and 145A>G in intron 10. Association analysis revealed four markers (114T>A, 94T>A, 460G>A, 719A>G) whose frequencies were significantly different in intermediate (enzyme activity or=140 ng/mL/h) methylators (p<0.001). Haplotype analysis found one haplotype to be associated with intermediate TPMT activity., Conclusions: Our results point to several markers that predict reduced enzyme activity. None of the identified markers were associated with high enzyme activity.

Thiopurine S-methyltransferase (TPMT) is a biotransformation phase II enzyme responsible for the metabolic inactivation of thiopurine drugs. The present study was carried out to investigate the inhibitory potential of 15 nonsteroidal anti-inflammatory drugs (NSAIDs) on human TPMT activity in vitro. TPMT activity was measured in pooled human erythrocytes in the absence and presence of various NSAIDs using the previously published high-performance liquid chromatography-UV method. To determine the inhibition type and K(i) value for each compound, we performed kinetic analysis at five different inhibitor concentrations close to the IC(50) value obtained in preliminary experiments. Naproxen (K(i) = 52 microM), mefenamic acid (K(i) = 39 microM), and tolfenamic acid (K(i) = 50 microM) inhibited TPMT activity in a noncompetitive manner. The estimated K(i) values for the inhibition of TPMT by ketoprofen (K(i) = 172 microM) and ibuprofen (K(i) = 1043 microM) indicated that the propionic acid derivatives were relatively weak inhibitors of TPMT. Our results suggest that coadministration of thiopurines and various NSAIDs may lead to drug interactions.

Data on the pharmacokinetics (PKs) of penicillin G (PEN) in neonates date back to the 1970s and do not include data for very-low-birth-weight (VLBW) neonates. The aim of this study was to describe the steady-state PKs and to establish an optimal regimen for the dosing of PEN in neonates with gestational ages of less than 28 weeks and birth weights of less than 1,200 g. Two PEN dosing regimens were studied: 50,000 IU (30 mg)/kg of body weight every 12 h (q12h) (group 1; n = 9) and 25,000 IU (15 mg)/kg q12h (group 2; n = 9). Samples for PK analysis were drawn before the injection of PEN and at 2 and 30 min and 1.5, 4, 8, and 12 h after intravenous injection of the third to eighth PEN doses. The PEN concentration was measured by a high-performance liquid chromatography with UV detection technique. The median peak and trough concentrations of PEN were 147 mug/ml (lower and upper quartiles, 109 and 157 mug/ml, respectively) and 7 mug/ml (lower and upper quartiles, 5 and 13 mug/ml, respectively) after administration of the dose of 50,000 IU and 59 mug/ml (lower and upper quartiles, 53 and 78 mug/ml, respectively) and 3 mug/ml (lower and upper quartiles, 3 and 4 mug/ml, respectively) after administration of the dose of 25,000 IU. The PEN half-life (median and lower and upper quartiles for group 1, 3.9 h and 3.3 and 7.0 h, respectively; median and lower and upper quartiles for group 2, 4.6 h and 3.8 and 5.6 h, respectively) was longer in VLBW neonates than in adults and term infants. PEN renal clearance correlated with creatinine clearance (R(2) = 0.309596; P = 0.038). Only a median of 34% (lower and upper quartiles, 28 and 37%, respectively) of the administered dose was excreted in urine within the following 12 h. We conclude that in VLBW infants a PEN dose of 25,000 IU (15 mg)/kg q12h is safe and sufficient to achieve serum concentrations above the MIC(90) for group B streptococci for the entire dosing interval.

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6 ng/ml/h. For heterozygous individuals (n = 18), TPMT activity was 48.1 +/- 11.7 ng/ml/h. Wild type individuals (n = 81) revealed significantly higher TPMT activity 79.3 +/- 20.7 ng/ml/h (P < 0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required.