Parkinson’s disease (PD) is a neurodegenerative disease characterized by α-synucleinopathy, which involves all districts of the brain-gut axis, including the central, autonomic and enteric nervous systems. Previous findings suggest that the intestinal microbiome is altered in PD and is related to motor phenotype. However how dysbiosis arises and whether this feature contributes to PD pathogenesis remains unknown. The aim was to evaluate gut microbiome and the serum cytokine profile in PD. We quantified serum interleukin (IL) levels (IL-1β, IL-6, IL-10, TNF-α, IFN-γ) in 55 PD patients. Study of the fecal samples was performed by real time PCR method and bacteriologically. Discovered the relationship between the intensity of dysbiosis and the level of proinflammatory cytokine IFN-γ, IL-6.We show that disturbances in plasma cytokine level could be more profound in PD patients with altered composition of intestinal microbiota, which may explain the mechanism of influence of microbiota composition on the PD manifestations.
Statement. We recently developed a new treatment approach for infant leukemia with MLL rearrangements (MLL-Baby) that consists of intermittent pulses of all-trans retinoic acid (ATRA) and conventional chemotherapy. Here, we present data on the molecular remission status of patients with the MLL/AF4 rearrangement enrolled in this study. Materials and methods. Six patients with MLL/AF4 rearrangement have been enrolled in the MLL-Baby study in Ekaterinburg and Moscow since September 2003. Median age was 6.5 months (range 1.5–10 months). All have had the BI immunophenotype. Leucocytes from bone marrow (BM) and peripheral blood (PB) were obtained. Quality and integrity of RNA was estimated using RNA 6000 Nano LabChip kit and 2100 Bioanalyzer (Agilent Technologies). Only RNA with RNA integrity number more than 4.5 was taken further. Qualitative PCR for MLL/AF4 fusion gene (FG) was performed either by nested PCR (4 patients) (A. Borkhardt et al, 1994) or by low-density biochips (2 patients). Conditions of qualitative real-time PCR (qRT-PCR) for MLL-AF4 FG detection were previously described (J. Gabert et al., 2003). β2-microglobulin was used as the control gene. Normalized copy number (NCN) of MLL/AF4 was calculated. Sensitivity of qRT-PCR for BM specimens was 1×10−4 – 6×10−5, for PB was 1×10−4 – 7×10−4. In accordance with the treatment design all patients were enrolled on the high risk (HR) schedule as approved by Local and Federal Ethics committees. Parents’ informed consent was obtained. Results. We found 2 types of MLL/AF4 FG clearance. In 4 patients the elimination rate of FG transcripts was very high. In this group FG could not be detected after 1 course of ATRA that followed induction therapy or after HR 1 (I); in 1 patient with MLL/AF4, FG total clearance was achieved after HR block 3 (VI) respectively; 1 patient currently under treatment has few copies of MLL/AF4 after HR block 2 (V). There was no correlation between the rate of elimination of MLL/AF4 FG with age, initial WBC count or type of MLL/AF4 transcript. Once achieved, the molecular remission in 5 patients has remained PCR negative. Conclusions. Achievement of molecular remission is one of the most important steps toward long-term survival and cure. Despite differences in the rate of elimination of MLL/AF4 FG, 5 of 6 patients treated on the MLL-Baby protocol have remained in hematological and molecular remission with a median follow-up 18 months (range 6–45 months). None have relapsed. Moreover, the youngest patient, a girl who was 2 months old at the time of diagnosis, has had the longest continuous molecular remission of up to 45 months. The 6th patient has single copies of MLL/AF4 after HR block 2 (V) and is currently receiving therapy.
Statement. The MLL-MLLT4 (former MLL-AF6) fusion gene (FG) is relatively rare genetic abnormality, predominantly found in AML. It averages 3–5% among other MLL rearrangements. Here we present data of MRD monitoring in 2 patients with AML carrying an MLL-MLLT4 rearrangement by 2 approaches: using FG transcript at RNA/cDNA level in comparison with FG at genomic DNA level. Materials and methods. Patients (pts) were diagnosed according French-American-Britain (FAB) classification. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics, FISH and reverse transcriptase PCR (RT-PCR). RT-PCR products were directly sequenced afterwards. In both cases identification of genomic chromosomal breakpoint sequences within MLL and MLLT4 genes was done by long-distance inverse PCR (LDI-PCR). MRD quantification in genomic DNA was performed using patient-specific primers and probes by real-time quantitative PCR (RQ-PCR). 500 ng of DNA was used per reaction. Standard curve was received by serial 10-fold dilutions of pts’ DNA into the DNA isolated from pooled lymphocytes of ten healthy donors. b-actin was used as DNA quality and quantity control. Detection of FG transcript kinetics during treatment was performed by RQ-PCR according to “Europe Against Cancer” recommendations for normalization by using control gene ABL (Gabert J. et al Leukemia, 2003, 17) and for using 10-fold dilutions of plasmids carrying MLL-MLLT4 fragment as source of standard curve. MRD value for cDNA targets were estimated as previously described (Beillard E. et al Leukemia, 2003, 17). Each sample was run in triplicates. According to the treatment design, time-points for MRD estimation were scheduled before each block of treatment. Totally 5 samples were evaluated in each patient (initial and 4 follow-ups). FLT3-ITD status was estimated at the time of diagnosis. Informed consent was obtained in both cases. Patients’ characteristics, treatment and clinical outcome Case # 1 Case #2 Age 58 13 Sex Male Male Initial WBC*106/ml 5.5 94.9 Immunophenotype CD34+CD117+HLA-DR+ CD11c+CD13+CD33+CD65+ CD34+CD117+CD13+ CD33+CD45+MPO+ Cytogenetics 46, XY, del(5)(q?), der(5)t(5;6;11) (q22;q15q27;q23), der(6)t(5;6) (q22;q15), del der(11) 46, XY, t(6;11)(q27;q23) FISH with LSI MLL MLL deletion MLL split RT-PCR MLL-MLLT4 positive MLL-MLLT4 positive MLL-MLLT4 FG transcript exon 9-exon 2 exon 9-exon 2 Localization of genomic chromosomal breakpoint within MLL and MLLT4 intron 9-intron 1 intron 9-intron 1 FLT3-ITD Negative Negative Induction treatment 7+3 AIE Consolidation therapy 2× HAM 2× HDAC 1× HAM 1× FLAG 1× HAE Maintenance − + Duration of therapy, months 8 7 Achievement of CR + + OS, months 8 7 EFS, months 6 5 Current status Alive in CR Alive in CR Results. Despite of achievement of CR, MLL-MLLT4 FG transcripts were detected in every sample tested after induction and consolidation chemotherapy by RQ-PCR. MRD value in case #1 in cDNA was fluctuated significantly within 2 log. Although in case #2 there was successive reduction from 260% at the beginning of treatment till 0.7% before maintenance therapy (after HAE block). Limited dilution series of a MLL-MLLT4-positive RNA into RNA of ten healthy donors showed a sensitivity limitation of 1E–05. For quantification of genomic chromosomal breakpoint sequences b-actin was amplified in each well. Deviation between Ct values of b-actin in different wells did not exceed ±2.0. In case # 1 the standard curve of the RQ-PCR assay for MLL-MLLT4 FG had slope of −3.19. Correlation coefficient was 0.987. Quantitative range of this assay was 1E–04 and sensitivity 1E–5. It was also observed a considerable variation of MRD levels in genomic DNA during treatment, like it was observed in MRD monitoring by FG transcripts. Fluctuations run up to 2.5 log. In case #2 the standard curve of the RQ-PCR assay for MLL-MLLT4 had a slope of −3.63 with correlation coefficient 0.992. Quantitative range of this assay was reached 1E–04 with sensitivity 1E–05. MRD level in this patient constantly decreased. Conclusions. The same tendency has been shown in each patient: fluctuations of MRD levels (2.5 log in case #1) and successive reduction (case #2). Results received at RNA/cDNA level and in genomic DNA cannot substitute each other, but they can be used as additives. It has been demonstrated that quantification of MLL-MLLT4 FG in genomic DNA is precise and suitable for MRD monitoring.
Lyme borreliosis (LB) is the most common transmissible disease of tick-borne origin. This disease is a significant medical challenge due to the damage to many organs and systems and the tendency to chronicity and long-term disability in the future. Currently, there is an upward trend in the incidence of Lyme disease in Sumy Oblast. Thus, in Ukraine in 2015 the incidence was 7.96 per 100,000 population, and in 2019 it increased to 10.62. The average level of indicators was exceeded in Kyiv Oblast (29.0), Cherkasy Oblast (25.4), Vinnytsia Oblast (23.09), Sumy (25.89) Oblast, and Kyiv (2.54). The peak of tick activity in Ukraine is registered in May and has increased by 4.23 times. The maximum number of patients falls within the working population and causes significant state financial damage. The increase in the number of cases indicates a spread of Lyme disease in Sumy Oblast. In order to prevent the spread, it is necessary to study all possible causes of this disease and find ways to eliminate them. This was the ground for the creation of a unified anonymous questionnaire, which covers the main issues regarding this disease from the standpoint of different population groups. A unified anonymous "Questionnaire for Lyme disease patients" contains 16 multiple choice questions and an option for comments. The first group of questions concerned gender, age, and social data of respondents (the largest part of patients fell within the working population – 42.86%). The next group of questions clarified the epidemiological features of this pathology (circumstances, date, time, location, body part bitten). Most often tick bites occurred during walks in the urban forests (42.86% of cases). Most respondents reported that tick bites had happened in June and July (39.29%). Other questions were designed to determine patients' awareness about Lyme borreliosis prevention. The next group of questions was aimed at clarifying the clinical features of the disease (lower extremities were the most frequent site of a tick bite) (46.43%), with erythema dominating among the clinical manifestations of Lyme borreliosis (91.07%). According to the anonymous questionnaire, we analyzed and evaluated awareness of diagnosis, clinical signs, and measures of Lyme disease prevention in the patients who were receiving treatment at Municipal Non-Commercial Enterprise of Sumy Regional Council "Medical Clinical Center of Infectious Diseases and Dermatology Named After Z. Krasovytskyi" and SSU University Clinic.
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