Andrzej TRETYN

INTRODUCTION: Cyclophosphamide (CTX) is an alkylating agent of the nitrogen mustard type, regarded as one of the most potent immunosuppressive drugs available. An activated form of this drug, phosphoramide mustard, alkylates, or binds, to DNA. Its cytotoxic effect is mainly due to cross-linking of strands of DNA and RNA, and to inhibition of protein synthesis. Its has been shown that CTX suppress T-helper cell functions with prolonged reduction of B cells due to the slower rate of recovery of B lymphocytes from an alkylating agent. As in many chemotherapeutic drugs, a major hindrance to the effectiveness of cyclophosphamide in long term leukemic therapy is the target cells subsequent development of resistance against the drug. OBJECTIVE: The main objective of the research was to implement the determinations of the ex vivo resistance to cyclophosphamide profile and to identify the genetic profile for pediatric patients with acute leukemias. METHODS: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix) for 51 patients with ALL and 16 patients with AML. Hierarchical clustering, assignment location and biological function were performed during the correlation analysis for identified probe sets. Verification of the relative expression level of selected genes was carried out by real time PCR. In order to gain new insights into the molecular mechanisms involved in cyclophosphamide resistance, we performed array-based comparative genomic hybridization (aCGH) on a series of 24 primary acute leukemias, using a SurePrint G3 Human CGH Microarray, 8x60K (Agilent). Data was analyzed by bioinformatics tools: Partek Genomics Suite, PANTHER tools, KEGG Pathway, Agilent Feature Extraction & CytoGenomics. RESULTS: Based on the global expression profile and LC50 values we found, that cyclophosphamide and bortezomid are demonstrating the most different resistances profile, in relation to 20 antileukemic drugs analyzed in the entire research. We observed a multitude of differentially expressed genes, e.g., AKR1C3 (Fold Change=2,81, p-value=0,049), ANXA1 (FC=3,04, p=0,011), BCL2A1 (FC=2.69, p=0,019), SERPINA1 (FC=2.12, p=0.014), DHRS7 (FC=2.13, p=0.005), PCDH9 (FC=-4.58, p=0.015), TTC28 (FC=-2.25, p=0.033) and DUSP1; (FC=-2.91, p=0,002). We assigned the differentially expressed genes to functional pathways (Table 1). Table 2 shows consistently amplified and deleted regions in acute leukemias samples. A 0.29-Mb stable deleted region involving 10 genes was detected at 10q23.31. Among the…

Purpose Besides conventional kidney diseases diagnostics, micro RNAs (miRNAs) assessment in urine and serum is considered to be a promising non-invasive method of diagnostics of renal parenchymal diseases and valuable therapeutic target also. The purpose of the study was to investigate the role of several miRNAs as a markers of kidney damage. Methods Assessment of 45 chronic kidney disease (CKD) patients stage 1–4 and 17 healthy control. Sample of urine and blood was taken from each participant for molecular analysis using Real Time PCR method to identify such micro-RNAs as: hsa-miR-155-5p, hsa-miR-214-3p, hsa-miR-200a-5p, hsa-miR-29a-5p, hsa-miR-21-5p, hsa-miR-93-5p, and hsa-miR-196a-5p. Basic biochemical test was done. Analysis was performed in CKD patients group and subgroup with chronic glomerulonephritis (CGN) confirmed by kidney biopsy. Moreover, analysis was performed in subgroup with different estimated glomerular filtration rate (eGFR) (according to CKD–EPI equation: eGFR &...

Introduction: The main objective was to implement the determinations of the ex vivo resistance to cyclophosphamide and to identify the genetic profile for pediatric patients with acute leukemias. Methods: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). We performed also array-based comparative genomic hybridization using a SurePrint G3 Human CGH Microarray. Data was analyzed by bioinformatics tools. Verification of the relative expression level of 20 genes was carried out by qRT- PCR. Results: We observed a multitude of differentially expressed genes, e.g. ANXA1 (FC=3,04), BCL2A1 (FC=2,69), SERPINA1 (FC=2,12), DHRS7 (FC=2,13), PCDH9 (FC=- 4,58), TTC28 (FC=-2,25) and DUSP1 (FC=-2,91). The expression of genes that code for inflammation mediated by chemokine and cytokine signaling, Wnt...

Here we determined the impact of salt shock and salt stress on the level of DNA methylation in selected CpG islands localized in promoters or first exons of sixteen salt-responsive genes in beets. Two subspecies differing in salt tolerance were subjected for analysis, a moderately salt-tolerant sugar beetBeta vulgaris ssp.vulgariscv. Huzar and a halophytic beet,Beta vulgaris ssp.maritima. The CpG island methylation status was determined. All target sequences were hyper- or hypomethylated under salt shock and/or salt stress in one or both beet subspecies. It was revealed that the genomic regions analyzed were highly methylated in both, the salt treated plants and untreated controls. Methylation of the target sequences changed in a salt-dependent manner, being affected by either one or both treatments. Under both shock and stress, the hypomethylation was a predominant response in sugar beet. InBeta vulgaris ssp.maritima, the hypermethylation occurred with higher frequency than hypomet...

The increase of human population and associated increasing demand for agricultural products lead to soil over-exploitation. Biofertilizers based on lyophilized plant material containing living plant growth-promoting microorganisms (PGPM) could be an alternative to conventional fertilizers that fits into sustainable agricultural technologies ideas. We aimed to: (1) assess the diversity of endophytic bacteria in sugar and sea beet roots and (2) determine the influence of osmoprotectants (trehalose and ectoine) addition during lyophilization on bacterial density, viability and salt tolerance. Microbiome diversity was assessed based on 16S rRNA amplicons sequencing, bacterial density and salt tolerance was evaluated in cultures, while bacterial viability was calculated by using fluorescence microscopy and flow cytometry. Here we show that plant genotype shapes its endophytic microbiome diversity and determines rhizosphere soil properties. Sea beet endophytic microbiome, consisting of ge...

Osteoarthritis (OA) is a degenerative disease of the joints, characterized by irreversible destruction of articular cartilage. The disease process is accompanied by changes of immunological nature, resulting in local inflammatory reactions, with the production of proinflammatory cytokines and metalloproteinases. There is currently no effective treatment resulting in repair of degraded cartilage. Clinical application of mesenchymal cells (MSCs) creates new possibilities in the treatment of incurable diseases. Multipotent MSCs exhibit immunosuppressive activity and limited immunogenicity and have the potential to differentiate in vitro towards adipocytes, osteocytes, chondrocytes, myocytes and endothelial cells. Thanks to these biological properties, they are increasingly used in clinical therapies. In few scientific papers, the safety of cellular therapies in the group of dogs diagnosed with OA has been confirmed. In patients undergoing treatment with autologous intra-articular injec...

INTRODUCTION: Resistance to drugs is connected with several genomic changes, but the major mutations are still unknown. Daunorubicin (DNR), idarubicin (IDA), doxorubicin (DOX) and mitoxantrone (MIT) are chemotherapeutics of the anthracycline family that are commonly used to treat acute leukemias (AL). OBJECTIVE: The aim of the study was to identify changes on the genome level and compare them with ex vivo resistance to DNR, IDA, DOX and MIT among children diagnosed with acute lymphoblastic (ALL) or myeloblastic (AML) leukemia. METHODS: The in vitro drug resistance profile was determined in MTT cytotoxicity assay, which was performed on mononuclear cells taken from 44 (aCGH) and 187 (qPCR) patients. The cyanine labeled genomic DNA (Cy3 - reference, Cy5 - patient) was hybridized to comparative genomic hybridization array (SurePrint G3 Human CGH Microarray 8x60K, Agilent). Data were analyzed using bioinformatics tools, like: Feature Extraction and CytoGenomics (Agilent) and databases, ...

INTRODUCTION: Busulfan (BUS) is bifunctional cell cycle non-specific alkylating antineoplastic agent. High-dose busulfan in combination with cyclophosphamide is an effective preparative regimen for patients undergoing allogeneic or autologous bone marrow transplantation for leukemias and solid tumors. The main mechanism of action involves the interference of DNA replication and RNA transcription (by alkylation and cross-linking of strands), which results in the disruption of nucleic acid functions. Alterations in drug transport and metabolism, increased activity of glutathione S-transferase and aldehyde dehydrogenase activity or enhanced DNA repair may also play a role in the resistance to this alkylator. OBJECTIVE: The aim of this study was to elucidate candidate genes and molecular pathways involved in ex vivo resistance to busufan in childhood acute leukemias. METHODS: In order to determine the in vitro BUS resistance profile, MTT cytotoxicity assay was performed on mononuclear c...

This is the first communication of micropropagation system for Inula germanica using seedling explants germinated in vitro. The development of this system gives the possibility of future reintroduction of I. germanica providing a way to stabilize or re-establish its population. Shoot tips and fragments of cotyledons, hypocotyls and roots were isolated from ten-day-old seedlings. Explants were put on MS medium containing 1.0 mg l-1 benzylaminopurine and 0.1 mg l-1 naphthaleneacetic acid and cultured under continuous white fluorescent light (45 μmol.m-2.s-1) at 26 ± 1 °C. The highest percentage of shoot organogenesis (83.3%) was recorded for hypocotyl, while the highest average number of shoots per explant (12.0) was recorded for shoot tips. In subsequent subcultures, multiplication rate decreased to 3.0-4.9 shoots per explant. Less than 19% shoots were able to root on the solid medium without auxins. The highest rooting efficiency (69.3%) was recorded for solid medium supplemented wi...

Background: COVID-19 vaccines induce a differentiated humoral and cellular response, and one of the comparable parameters of the vaccine response is the determination of IgG antibodies. Materials and Methods: Concentrations of IgG anti-SARS-CoV-2 antibodies were analyzed at three time points (at the beginning of May, at the end of June and at the end of September). Serum samples were obtained from 954 employees of the Nicolaus Copernicus University in Toruń (a total of three samples each were obtained from 511 vaccinated participants). IgG antibody concentrations were determined by enzyme immunoassay. The statistical analysis included comparisons between vaccines, between convalescents and COVID-19 non-patients, between individual measurements and included the gender, age and blood groups of participants. Results: There were significant differences in antibody levels between mRNA and vector vaccines. People vaccinated with mRNA-1273 achieved the highest levels of antibodies, regardl...

2747 Poster Board II-723 Background: A distinct phenotype of combined ex vivo drug resistance and gene expression associated with this phenotype discriminates treatment outcome and identifies a subset of patients with a markedly inferior outcome in childhood acute lymphoblastic leukemia (ALL). No similar relationship was found so far in acute myeloblastic leukemia (AML). We hypothesized that drug sensitivity profile combined with gene expression profile can provide a new insight into selection of drugs used in high-dose therapy before hematopoietic stem cell transplantation and determine the role of specific drugs. Objective: The aim of the study was to analyze the gene expression profile in correlation to the ex vivo obtained chemosensitivity profile of drugs used in high-dose therapy before hematopoietic stem cell transplantation in children with ALL and AML. Methods: We tested leukemic cells from 56 children (43 ALL de novo, 8 relapsed ALL, 5 AML de novo) for ex vivo sensitivity ...

Background: The presented research made it possible to obtain the characteristics of changes in anti-SARS-CoV-2 IgG within one year of vaccination in healthcare workers. Materials and Methods: The research group consisted of 18,610 participants represented by medical and administration staff. IgG antibody concentrations were determined by ELISA. Results: At 5–8 months after full vaccination, the levels of anti-SARS-CoV-2 IgG with equal vaccines were similar. The exception was JNJ-78436735, for which IgG levels were significantly lower. In the 9th month after vaccination, an increase in the anti-SARS-CoV-2 IgG level, suggesting asymptomatic infection, was observed in a large group of participants. Significantly higher levels of anti-SARS-CoV-2 IgG antibodies were observed after the booster dose compared to the second dose. The increase in antibodies was observed already around the 5th day after the injection of the booster dose, and was maximized at approximately the 14th day. Conclu...

At the end of 2020, population-based vaccination programs with new generation mRNA-based vaccines began almost all over the world. The aim of the study was to evaluate the titer of anti-SARS-CoV-2 IgG antibodies against the S1 subunit of the virus’s spike protein as a marker of the humoral response in 477 patients and the concentration of interferon-gamma as an indicator of cellular response in 28 individuals. In our studies, we used serological enzyme-linked immunosorbent assays. IgG was measured in weeks 2 and 3 after the first dose and 1–5 weeks after the second dose of an mRNA vaccine in seropositive and seronegative individuals as well as in symptomatic and asymptomatic convalescents. High levels of antibodies were observed in 98% of our vaccinated cohort, and the presence of protective T cells was confirmed in the blood samples of all participants. The humoral immune response is diversified and is visible as early as 2–3 weeks after the first dose of the mRNA vaccine. The leve...

Summary Plants have developed mechanisms to integrate both endogenous and environmental cues for regulation of flowering time. When environmental and physiological (e.g. photoperiod, temperature) (e.g. stage of development) conditions are appropriate plants undergo the floral transition and become reproductive. The timing of flowering initiation depends on the balanced expression of many different genes that are regulated by both endogenous and environmental factors. As a result of physiological, genetic, and molecular analysis of Arabidopsis thaliana mutants altered in flowering time the existence of a long-promotion pathway, a gibberellic-acid promotion pathway, as well as vernalization and autonomous pathway were discovered and characterized. A few dozen of genes invilved in flower induction of Arabidopsis were identified. Some of them can integrate two or three flowering pathways. Floral repression is likely to be the principal mechanism for maintaining vegetative development. Floral repressor inhibit the floral signaling pathways at various levels. Some of genes involved in vernalization and photoperiodic flower induction encode putative chromatin-associated proteins. They probably function as epigenetic silencers that repress promotion of flowering, and thereby maintain vegetative growth. The complete genome sequences of two plant species; Arabidopsis thaliana (long-day dicot) and Oryza sativa (short-day monocot) have been published recently. Since that time, comparative genomics andmolecular genetics on photoperiod-induced flowering process became possible. Using this approach some differences between long- and short-day plants were established at the molecular level.

ABSTRACT Zaburzenia mechanizmów odpowiedzi i naprawy uszkodzeń DNA powodują wzrost niestabilności genomu i predysponują do nowotworów. Krytyczną rolę w utrzymaniu stabilności genomu odgrywają geny TP53 i BRCA1/2, których mutacje germinalne predysponują do nowotworów piersi i jajnika. Białka BRCA1/2 są znanymi czynnikami podatności na nowotwory piersi, które biorą udział w utrzymanie stabilności genomu poprzez zaangażowanie w procesy naprawy dwuniciowych pęknięć DNA na drodze rekombinacji homologicznej. Białko p53, produkt genu TP53, określane jako „strażnik genomu” odpowiedzialne jest za utrzymanie integralności genomu uczestnicząc w mechanizmach odpowiedzi na uszkodzenie DNA takich jak: zatrzymanie cyklu komórkowego, indukcja procesów naprawy DNA lub apoptozy w przypadku gdy uszkodzenia nie mogą być skutecznie naprawione. p53 i BRCA1/2 dzięki swoim funkcjom należą do jednych z najszerzej badanych białek ludzkich. Niemniej homologi białek BRCA1/2 są także obecne u roślin wyższych. Analizy roślinnych i zwierzęcych białek BRCA1/2 ujawniają wysoką homologię w regionach domen odpowiedzialnych za zakonserwowane funkcje tych białek. Do tej pory badania funkcji BRCA1/2 u roślin przeprowadzono wyłącznie na rzodkiewniku pospolitym (A thaliana). W genomie tej rośliny odkryto dwa geny homologiczne do BRCA2 (AtBRCA2), których produkty białkowe podobnie jak u zwierząt uczestniczą w reperacji dwuniciowych pęknięć na drodze rekombinacji homologicznej, a także odgrywają ważną rolę w rekombinacji mejotycznej. Z kolei pojedynczy homolog BRCA1 obecny u rzodkiewnika (AtBRCA1) uczestniczy w kontroli cyklu komórkowego i reperacji DNA. Mutanty roślinne w genach BRCA1/2 wykazują wrażliwość na substancje powodujące uszkodzenia DNA reperowane drogą rekombinacji homologicznej. Dotychczasowe wyniki badań wskazują na funkcjonalną konserwację genów BRCA1/2 u roślin. W odróżnieniu do genów z rodziny BRCA, homologii białka p53 nie zostały zidentyfikowane u roślin co wskazuje, że jeśli istnieje funkcjonalny roślinny odpowiednik białka p53 to nie dzieli on zakonserwowanych sekwencji ze zwierzęcymi p53.

An efficient shoot propagation system for Carlina acaulis was developed in this study. The experimental mate- rial consisted of shoot tips and fragments of hypocotyls excised from 10-day-old seedlings. The explants were transferred to proliferation medium supplemented with different types of cytokinins: 6-benzylaminopurine (BA, 4.4 or 13.3 μM), kinetin (Kn, 4.7 or 13.9 μM) and zeatin (Zea, 4.6 or 13.7 μM) in combination with naphthale- neacetic acid (0.54 μM NAA). The morphogenetic response was best in culture on medium supplemented with 13.3 μM BA, and shoot organogenesis frequency was highest for shoot tips (100%). On average, 7.5 shoots were induced per explant of the initial material, and the multiplication rate in five subsequent subcultures was 6.1. Shoot length was lower in culture with BA in the medium than with Kn or Zea. Plantlets rooted with 60% fre- quency in vitro on full-strength MS medium and with 55.3% frequency ex vitro. Reduction of the mineral salt concentration (...

The aim of the presented research was to examine the morphogenetic response of Polemonium coeruleum explants. The donor material were 10-day-old seedlings. Surface sterilized seeds were germinated on MS medium supplemented with GA3 (1 mg©dm-3). Seedling explants (shoot tips, fragments of cotyledons, hipocotyls and roots) were isolated and transferred onto solidified MS medium supplemented with different types of cytokinins (BA, KN, ZEA, 2iP) at concentrations 1.0, 3.0 and 5.0 mg©dm-3 in combination with NAA (0.1 mg©dm-3). All explant types were characterized by callus proliferation. It was observed that calli developed on the entire surface of hipocotyl and root fragments. On the other hand, shoot tips and cotyledonary petioles formed callus tissue at the cut ends, and petioles only at abaxial ends. The growth of calli on all explant types was strongly stimulated by ZEA. Among the explants tested, only shoot tips exhibited shoot organogenesis. The highest frequency of shoot organoge...

Arabidopsisroot system responds to phosphorus (P) deficiency by decreasing primary root elongation and developing abundant lateral roots. Feeding plants with ascorbic acid (ASC) stimulated primary root elongation in seedlings grown under limiting P concentration. However, at high P, ASC inhibited root growth. Seedlings of ascorbate-deficient mutant(vtc1)formed short roots irrespective of P availability. P-starved plants accumulated less ascorbate in primary root tips than those grown under high P. ASC-treatment stimulated cell divisions in root tips of seedlings grown at low P. At high P concentrations ASC decreased the number of mitotic cells in the root tips. The lateral root density in seedlings grown under P deficiency was decreased by ASC treatments. At high P, this parameter was not affected by ASC-supplementation.vtc1mutant exhibited increased lateral root formation on either, P-deficient or P-sufficient medium. Irrespective of P availability, high ASC concentrations reduced ...

The impact of interleukin 28B (IL-28B) on the results of interferon (IFN)-based therapy in patients chronically infected with hepatitis B virus (HBV) is poorly understood. The aim of this study was to evaluate the relationship between IL-28B markers and the response to IFN monotherapy in Polish patients with anti-hepatitis B e (HBe)-positive chronic hepatitis B (CHB). We determined three single-nucleotide polymorphisms (SNPs) of IL-28B (rs12979860, rs12980275, and rs8099917) in 86 patients who were treated with pegylated interferon (PEG-IFN) for 48 weeks. The effectiveness of the therapy was evaluated based on the virological and biochemical response. The primary efficacy parameters were the HBV DNA viral load below 400 IU/ml and 2,000 IU/ml in combination with alanine aminotransferase (ALT) normalization (<40 IU/l), measured 24 weeks after the treatment. Viral load below 400 IU/ml or 2,000 IU/ml with ALT normalization was achieved by 37 % and 46 % of patients, respectively. It h...

Regenerated plants of Carlina acaulis subsp. simplex induced on shoot tips and fragments of hypocotyls, cotyledons and roots were used as an experimental material. Explants were isolated from 10-day-old, sterile seedlings and were put on growth media supplemented with BA (3 mg×dm-3), and NAA (0,1 mg×dm-3). Plantlets were acclimatized to ex vitro conditions and planted to the field. Analysis of flowering ability, inflorescence stem morphology, and survival level was the objective of the study. The plants regenerated from shoot tips and cotyledons were able to flower in the first year after acclimatization, however no vital seeds were found, while in the case of hypocotyl- and root- regenerated plants flowering appeared in the second year after acclimatization. Number of flowering-able plants grew in time, reaching 100% level. Few percent of inflorescence stems displayed branches ending with additional capitula. The number of this type of plants decreased in successive years, while th...

The prerequisite for shoot, root or somatic embryo formation in plant in vitro culture is the development of meristem from dedifferentiated cells of the explant tissue. Auxin and cytokinin levels and their relative ratios play a decisive role in inducing the morphogenetic pathways leading to shoot, root or somatic embryo formation in plant in vitro cultures. Exogenous auxin is required to maintain the high rate of an unorganised growth in plant cell suspension cultures. On the other hand, the proliferation of hairy root cultures is usually dependent on endogenous hormonal factors. Auxin and cytokinin execute their regulatory role by being involved in a cross-talk with numerous endogenous factors affecting cell division and differentiation. Among them, ascorbate/dehydroascorbate (ASC/DHA), glutathione/glutathione disulphide (GSH/GSSG) redox pair, H2O2 and other components of cellular redox systems play an important role in triggering developmental responses in plant in vitro culture. Ascorbate, glutathione and related enzymes participate in the responses to auxin/cytokinin treatments. In addition, they can even directly affect hormone metabolism in tissue. Ascorbate and glutathione have important regulatory roles in the process of cell-cycle progression within the meristems, where they participate in redox-dependent determination of proliferation and quiescence patterns. The mechanism underlying the regulatory effects of ascorbate and glutathione in cell divisions is not fully elucidated; however, it seems to be related to the regulation of nucleotide synthesis. Ascorbate ­levels in apoplast modulate the rate of organ elongation by increasing cell wall extensibility. Besides the effects on cell proliferation and growth, ascorbate and glutathione concentrations as well as the enzymes of their metabolism protect the in vitro cultured tissues against oxidative stress. This function is of particular importance during root regeneration and the elicitation of metabolite production by hairy root cultures, where increased levels of oxidising agents are often required to stimulate both processes. In this review, we report recent studies on the involvement of ascorbate and glutathione in the processes of regeneration and proliferation in plant tissue culture.

ABSTRACT The effect of two different iron chelates and iron concentration on multiplication, shoot growth, chlorophyll content and rooting of Carlina onopordifolia were studied in in vitro culture. FeEDTA presented in MS basal medium was replaced by FeEDDHA, which was applied in three concentrations: 93.5, 187.0 and 280.5 mg dm−3 (5.6 mg dm−3, 11.2 and 16.8 mg dm−3 Fe ions, respectively). Changing chelate or iron concentration in the medium had no effect on axillary shoot number proliferation, but growth of shoots was significantly inhibited by a two- and three-fold increase in concentration of FeEDDHA in the medium. Supplementation of the medium with FeEDDHA as Fe source significantly increased the level of chlorophyll in the leaves. After treatment of shoots with IBA for 5 s and growing them on the MS medium supplemented with FeEDTA, the number of roots per shoot was significantly higher than on medium containing FeEDDHA. Increasing the concentration of Fe ions in the medium after a short pulse (5 s) of IBA had no effect on shoot rooting. After 30 s of 1-g dm−3 IBA treatment, growth of roots on medium with FeEDDHA was stimulated. The survival rate was relatively low and did not depend on the type and concentration of iron chelate in the rooting medium.