Psychopharmacological and antibiotic drugs influence the genomic DNA methylation in SH-SY5Y neuroblastoma cells

Epigenetische Mechanismen wie DNA-Methylierung spielen bei zahlreichen physiologischen Prozessen, insbesondere bei der Regulation von Genexpression und Stabilisierung der Chromatinstruktur, eine wichtige Rolle. Obwohl Veränderungen in den Methylierungsmustern bei einer Vielzahl psychiatrischer Erkrankungen bekannt sind, ist der epigenetische Einfluss von Medikamenten speziell von Psychopharmaka bis heute kaum untersucht. Ziel dieser Arbeit war es deshalb den Einfluss von Mood-Stabilizern (Carbamazepin, Lithium, Valproinsäure), Neuroleptika (Haloperidol, Olanzapin, Quetiapin, Risperidon), Antidepressiva (Amitriptylin, Fluoxetin, Tranylcypromin) und den beiden Antibiotika Cefotiam und Ciprofloxacin auf die genomische DNA-Methylierung in Zellkultur zu prüfen. Als Referenzsubstanzen dienten 5-Aza-2’-deoxycytidin, Methotrexat und S-Adenosyl-L-methionin. Methoden: In Zellkulturexperimenten wurden SH-SY5Y Neuroblastomzellen mit den oben genannten Substanzen über 6 Stunden, 12 Stunden, 24 Stunden, 2 Tage und 5 Tage stimuliert, um Methylierungsveränderungen im zeitlichen Verlauf zu erfassen. Geeignete Stimulations-konzentrationen der einzelnen Substanzen wurden mit Hilfe eines MTT-Zellvitalitätstests bestimmt. Die Messung der genomischen Methylierung erfolgte mit einem modifizierten, fluoreszenzmarkierten Cytosin-Elongationsassay, beruhend auf einem DNA-Restriktionsverdau und einer anschließenden Fluoreszenzmarkierung mittels einer Elongationsreaktion. Ergebnisse: Bei den Referenzsubstanzen zeigte sich nach Behandlung mit 5-Aza-2’-deoxycytidin eine signifikante, nahezu vollständige Demethylierung nach zwei Tagen (p = 0,024) und auch bei Stimulation mit Methotrexat kam es innerhalb dieser Zeit zu einem signifikanten Abfall der Methylierung (p = 0,005). Hingegen hatte die Stimulation mit S-Adenosyl-L-methionin in den durchgeführten Versuchen keinen signifikanten Effekt auf die genomische DNA-Methylierung (p = 0,140). Die untersuchten Mood-Stabilizer Lithium (p = 0,007) und Valproinsäure (p = 0,030) bewirkten eine signifikante Hypomethylierung, wobei unter Valproinsäure diese Reaktion bereits innerhalb der ersten Stunden zu beobachten war, während bei Lithium ein vergleichbarer Abfall der Methylierung nach 12 Stunden einsetzte. Die mit Carbamazepin behandelten Zellen zeigten erst nach fünf Tagen eine Tendenz zur Hypomethylierung, die aber nicht signifikant war (p = 0,084). Bei den Neuroleptika hatte lediglich Risperidon einen signifikanten Einfluss im Sinne einer verminderten DNA-Methylierung innerhalb der ersten zwei Tage (p = 0,006). Zwei weitere getestete, atypische Neuroleptika Olanzapin (p = 0,246) und Quetiapin (p = 0,159) wiesen ebenfalls die Tendenz zur Demethylierung auf, wenngleich dies keine Signifikanz erreichte. Haloperidol als typisches Neuroleptikum veränderte während des gesamten Versuchsverlaufs die genomische Methylierung nicht signifikant (p = 0,437). Von den untersuchten Antidepressiva hatten sowohl das Trizyklikum Amitriptylin (p < 0,001) als auch das SSRI Fluoxetin (p < 0,001) eine hoch signifikante, hypomethylierende Wirkung; der MAO-Inhibitor Tranylcypromin beeinflusste den Grad der DNA-Methylierung nicht signifikant (p = 0,183). Die Antibiotika Cefotiam (p = 0,021) und Ciprofloxacin (p = 0,193) führten zu einer DNA-Hypermethylierung, die allerdings nur bei Cefotiam ein signifikantes Niveau erreichte. Die Ergebnisse belegen, dass einige Psychopharmaka insbesondere Mood-Stabilizer, Antidepressiva und in geringerem Ausmaß auch Neuroleptika epigenetische Veränderungen in Form einer verringerten DNA-Methylierung bewirken können. Im Hinblick auf mögliche pathologische Methylierungsmuster bei der Entstehung psychiatrischer Erkrankungen eröffnet dies neue Therapieansätze und sollte daher weiter untersucht werden. Außerdem sind zusätzliche genspezifische Analysen zum Einfluss von Psychopharmaka auf die DNA-Methylierung (z.B. Dopamintransporter, serotonerge Gene) notwendig, um möglicherweise pathophysiologische Zusammenhänge zu detektieren.Epigenetic mechanisms like DNA methylation are important for many physiological processes especially for regulation of gene transcription and stabilization of chromatin structure. Even though changes in the methylation pattern are known in many psychiatric diseases the epigenetic influence of medications especially psychotropic drugs has hardly been tested. Therefore the aim of this study was to examine the effects of mood stabilizers (carbamazepine, lithium, valproate), neuroleptics (haloperidol, olanzapin, quetiapine, risperidone), antidepressants (amitriptyline, fluoxetine, tranylcypromine) as well as the two antibiotics cefotiam and ciprofloxacin on genomic DNA methylation in cell culture. 5-Aza-2’-deoxycytidine, methotrexate and S-Adenosyl-L-methionine served as reference substances. Methods: In cell culture SH-SY5Y neuroblastoma cells were stimulated with the substances mentioned above for varying durations of 6 hours, 12 hours, 24 hours, 2 days and 5 days to quantify alterations in genomic methylation. The appropriate substance concentrations for stimulation were determined using a MTT cell viability assay. Genomic DNA methylation was measured by a modified, fluorescent Cytosin elongation assay based on a DNA restriction digestion and subsequent fluorescence labelling using an elongation reaction. Results: Treatment with the reference substance 5-Aza-2’-deoxycitidine showed a significant, nearly complete demethylation after two days (p = 0,024) and likewise stimulation with methotrexate lead to a significant decline of methylation (p = 0,005) within the same time. Stimulation with S-Adenosyl-L-methionine had no significant effect on genomic DNA methylation in the performed tests (p = 0,140). The investigated mood stabilizers lithium (p = 0,007) and valproate (p = 0,030) caused a significant hypomethylation. When using valproate hypomethylation set in within the first hours, whereas when using lithium a comparable decrease of methylation began after 12 hours. Cells treated with carbamazepine did not show a tendency towards hypometylation until the fifth day which was not significant (p = 0,084). Among the neuroleptics merely risperidone had a significant influence in terms of a reduced DNA methylation within the first two days (p = 0,006). Two more examined atypical neurolepics olanzapine (p = 0,246) and quetiapine (p = 0,159) indicated a tendency towards demethylation although to no significant level. Haloperidol as a conventional neuroleptic had no significant impact on genomic methylation during the whole experiment (p = 0,437). Among the tested antidepressants the tricyclic antidepressant amitriptyline (p < 0,001) as well as the SSRI fluoxetine (p < 0,001) showed highly significant hypomethylating properties; the MAO inhibitor tranylcypromine however did not significantly affect the degree of DNA methylation (p = 0,183). The antibiotics cefotiam (p = 0,021) and ciprofloxacin (p = 0,193) lead to DNA hypermethylation, though only cefotiam showed significant effects. These findings indicate that several psychotropic drugs especially mood stabilizers, antidepressants and to a lesser extent neuroleptics bring about epigenetic changes in the form of lowered DNA methylation. With regard to potential pathological methylation patterns in the development of psychiatric diseases this could disclose new therapeutic approaches and therefore should be further investigated. Additional gene specific analyses regarding the impact of psychotropic drugs on DNA methylation e.g. dopamine transporter and serotonergic genes are necessary to detect possible pathophysiological correlations

Risk factors and mortality in invasive Rasamsonia spp. infection: Analysis of cases in the FungiScope registry and from the literature

Background The new Rasamsonia spp. complex can develop invasive infection in immunosuppression or chronic pulmonary disease. It has potential to be misidentified as other genera due to morphological similarities. Nowadays, there is a gap of knowledge on this fungi. Objectives To provide knowledge base of risk factors and therapeutic decisions in invasive Rasamsonia spp. complex infection. Patients/Methods Cases of invasive infection due to Rasamsonia spp. (formerly Geosmithia/Penicillium spp.) from FungiScope registry and all reported cases from a literature were included. Results We identified 23 invasive infections due to Rasamsonia spp., six (26.1%) in the FungiScope registry. Main risk factors were chronic granulomatous disease (n = 12, 52.2%), immunosuppressive treatment (n = 10, 43.5%), haematopoietic stem cell transplantation (n = 7, 30.4%), graft-versus-host disease and major surgery (n = 4, 17.4%, each). Predominantly affected organs were the lungs (n = 21, 91.3%), disease disseminated in seven cases (30.4%). Fungal misidentification occurred in 47.8% (n = 11), and sequencing was used in 69.6% of the patients (n = 16) to diagnose. Breakthrough infection occurred in 13 patients (56.5%). All patients received antifungal treatment, mostly posaconazole (n = 11), caspofungin (n = 10) or voriconazole (n = 9). Combination therapy was administered in 13 patients (56.5%). Susceptibility testing showed high minimum inhibitory concentrations for azoles and amphotericin B, but not for echinocandins. No preferable treatment influencing favourable outcome was identified. Overall mortality was 39% (n = 9). Conclusion Rasamsonia spp. are emerging fungi causing life-threatening infections, especially in immunocompromised and critically ill patients. Mortality is high. Treatment is challenging and clinicians dealing with this patient population should become aware of this infection constituting a medical emergency

A Comparison of Aspergillus and Mucorales PCR Testing of Different Bronchoalveolar Lavage Fluid Fractions from Patients with Suspected Invasive Pulmonary Fungal Disease

In patients with hematological malignancies, bronchoalveolar lavage fluid (BALF) specimens are commonly used for the diagnosis of mold infections. However, it is not clear whether the cell pellet (P) or the supernatant fraction (S) of the BALF specimen is optimal for molecular diagnostic testing. Thus, 99 BALF specimens were collected from 96 hematology patients with or without allogeneic hematopoietic stem cell transplant. The cell pellets and supernatants were processed alone and in combination (S/P) for testing by two fungus-specific real-time PCR assays compliant with international recommendations. The results achieved with S/P were revealed to be superior in comparison to those achieved with S and P alone, with the use of each single fraction showing a reduced sensitivity for the detection of Aspergillus DNA (82% and 43% for S and P, respectively). In 57% of the samples, testing of the combination of S and P generated a lower quantification cycle value than testing of S or P alone. Molds would have been missed in 5 and 16 out of 28 samples if only S or P, respectively, was analyzed. No sample was positive by testing of S or P only. Similar results were obtained for the detection of Mucorales DNA in BALF specimens (reduced sensitivity of 67% and 50% for S and P, respectively). Study patients were categorized according to the current European Organization for the Research and Treatment of Cancer/Mycoses Study Group classification for invasive fungal disease (IFD), revealing that 35 patients had proven/probable IFD (36%), 47 patients had possible IFD (49%), and 14 patients had undetermined IFD (15%)

Matched-paired analysis of patients treated for invasive mucormycosis: standard treatment versus posaconazole new formulations (MoveOn)

International audienceBackground : First-line antifungal treatment for invasive mucormycosis (IM) consists of liposomal amphotericin B. Salvage treatment options are limited and often based on posaconazole oral suspension. With the approval of posaconazole new formulations, patients could benefit from improved pharmacokinetics, safety and tolerability.Objectives: Our aim was to assess the effectiveness of posaconazole new formulations for IM treatment.Methods : We performed a case-matched analysis with proven or probable IM patients from the FungiScope® Registry. First-line posaconazole new formulations (1st-POSnew) and first-line amphotericin B plus posaconazole new formulations (1st-AMB+POSnew) cases were matched with first-line amphotericin B-based (1st-AMB) treatment controls. Salvage posaconazole new formulations (SAL-POSnew) cases were matched with salvage posaconazole oral suspension (SAL-POSsusp) controls. Each case was matched with up to three controls (based on severity, haematological/oncological malignancy, surgery and/or renal dysfunction).Results : Five patients receiving 1st-POSnew, 18 receiving 1st-AMB+POSnew and 22 receiving SAL-POSnew were identified. By day 42, a favourable response was reported for 80.0% (n = 4/5) of patients receiving 1st-POSnew, for 27.8% (n = 5/18) receiving 1st-AMB+POSnew and for 50.0% (n = 11/22) receiving SAL-POSnew. Day 42 all-cause mortality of patients receiving posaconazole new formulations was lower compared with controls [20.0% (n = 1/5) in 1st-POSnew versus 53.3% (n = 8/15) in 1st-AMB; 33.3% (n = 6/18) in 1st-AMB+POSnew versus 52.0% (n = 26/50) in 1st-AMB; and 0.0% (n = 0/22) in SAL-POSnew versus 4.4% (n = 2/45) in SAL-POSsusp].Conclusions : Posaconazole new formulations were effective in terms of treatment response and associated mortality of IM. While posaconazole new formulations may be an alternative for treatment of IM, the limited sample size of our study calls for a cautious interpretation of these observations

Needles in a haystack: Extremely rare invasive fungal infections reported in FungiScopeⓇ—Global Registry for Emerging Fungal Infections

International audienc