Release of adenosine-induced immunosuppression: Comprehensive characterization of dual A2A/A2B receptor antagonist.

Immunosuppression is one of the main mechanisms facilitating tumor expansion. It may be driven by immune checkpoint protein expression, anti-inflammatory cytokine secretion or enhanced metabolic enzyme production, leading to the subsequent build-up of metabolites such as adenosine. Under physiological conditions, adenosine prevents the development of tissue damage resulting from a prolonged immune response; the same mechanism might be employed by tumor tissue to promote immunosuppression. Immune cells expressing A2A and A2B adenosine receptors present in an adenosine-rich environment have suppressed effector functions, such as cytotoxicity, proinflammatory cytokine release, antigen presentation and others, making them inert to cancer cells. This study was designed to investigate the dual antagonist potential of SEL330-639 to abolish adenosine-driven immunosuppression. SEL330-639 has slow dissociation kinetics. It inhibits cAMP production in human CD4+ cells, CD8+ cells and moDCs, which leads to diminished CREB phosphorylation and restoration of antitumor cytokine production (IL-2, TNFα, IL-12) in multiple primary human immune cells. The aforementioned results were additionally validated by gene expression analysis and functional assays in which NK cell line cytotoxicity was recovered by SEL330-639. Adenosine-driven immunosuppression is believed to preclude the effectiveness of immune checkpoint inhibitor therapies. Hence, there is an urgent need to develop new immuno-oncological strategies. Here, we comprehensively characterize SEL330-639, a novel dual A2A/A2B receptor antagonist effective in both lymphoid and myeloid cell populations with nanomolar potency. Due to its tight binding to the A2A and A2B receptors, this binding is sustained even at high adenosine concentrations mimicking the upper limit of the range of adenosine levels observed in the tumor microenvironment.

5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leucine zipper kinase inhibitors.

Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes and displays increased levels of expression in numerous cancer classes (colon, breast, brain, ovary, prostate and lung). Although no selective MELK inhibitors have yet been approved, increasing evidence suggest that inhibition of MELK would constitute a promising approach for cancer therapy. A weak high-throughput screening hit (17, IC50 ≈ 5 µM) with lead-like properties was optimized for MELK inhibition. The early identification of a plausible binding mode by molecular modeling offered guidance in the choice of modifications towards compound 52 which displayed a 98 nM IC50. A good selectivity profile was achieved for a representative member of the series (29) in a 486 protein kinase panel. Future elaboration of 52 has the potential to deliver compounds for further development with chemotherapeutic aims.

Analysis of genes involved in response to doxorubicin and a GD2 ganglioside-specific 14G2a monoclonal antibody in IMR-32 human neuroblastoma cells.

Neuroblastoma is the most common extra-cranial solid tumor of childhood and it is characterized by the presence of a glycosphingolipid, GD2 ganglioside. Monoclonal antibodies targeting the antigen are currently tested in clinical trials. Additionally, several research groups reported results revealing that ganglioside-specific antibodies can affect cellular signaling and cause direct cytotoxicity against tumor cells. To shed more light on gene expression signatures of tumor cells, we used microarrays to analyze changes of transcriptome in IMR-32 human neuroblastoma cell cultures treated with doxorubicin (DOX) or a mouse monoclonal antibody binding to GD2 ganglioside 14G2a (mAb) for 24 h. The obtained results highlight that disparate cellular pathways are regulated by doxorubicin and 14G2a. Next, we used RT-PCR to verify mRNA levels of selected DOX-responsive genes such as RPS27L, PPM1D, SESN1, CDKN1A, TNFSF10B, and 14G2a-responsive genes such as SVIL, JUN, RASSF6, TLX2, ID1. Then, we applied western blot and analyzed levels of RPS27L, PPM1D, sestrin 1 proteins after DOX-treatment. Additionally, we aimed to measure effects of doxorubicin and topotecan (TPT) and 14G2a on expression of a novel human NDUFAF2 gene encoding for mimitin protein (MYC-induced mitochondrial protein) and correlate it with expression of the MYCN gene. We showed that expression of both genes was concomitantly decreased in the 14G2a-treated IMR-32 cells after 24 h and 48 h. Our results extend knowledge on gene expression profiles after application of DOX and 14G2a in our model and reveal promising candidates for further research aimed at finding novel anti-neuroblastoma targets.

Ventilator-associated pneumonia monitoring according to the INICC project at one centre.

BACKGROUND: Pneumonia is a common complication of hospitalisation in severely ill patients who need mechanical ventilation. The aim of this study was to assess the usefulness of the International Nosocomial Infection Control Consortium programme for the surveillance of ventilator-associated pneumonia (VAP). METHODS: A prospective study (1 Jan 2012-30 June 2014) was conducted in the 20-bed ICU. The device utilisation ratios for lung ventilation and the frequency (density and incidence) and aetiology of VAP were estimated in ICU patients. RESULTS: From a total of 1097 patients, VAP infections were diagnosed in 93. Thirty percent of patients with VAP died. The incidence index was 8.47 per 100 admissions to the ICU. VAP infections accounted for 46% of the overall count of device-associated healthcare-associated infections. Mechanical ventilation was used in 71 ± 8 patients during the 11 862 patient days and 8425 ventilation days. The rate of VAP per 1000 ventilator days was 11.15/9.34 /10.23 in years 2012/2013/2014 (half a year), respectively. The main VAP pathogens were Acinetobacter baumannii (45%) and Pseudomonas aeruginosa (17%). CONCLUSION: During the reported time span, the incidence of VAP was lower than in the INICC report (2007-2012), but it was tenfold higher than in the NHSN/CDC report (dated 2012). Because of the unchanged VAP level during the 2.5-year observation period, the root cause needs to be determined and action should be taken to resolve this issue.

Monocyte chemoattractant protein-induced protein 1 impairs adipogenesis in 3T3-L1 cells.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1) encoded by the ZC3H12a gene (also known as Regnase-1) is involved in the regulation of degradation of mRNA of inflammatory modulators and for processing of pre-miRNA. These functions depend on the presence of the PIN domain. Moreover, MCPIP1 was described as a negative regulator of NF-κB and AP-1 signaling pathways although mechanisms underlying such activity remain unknown. We aimed at determining the role of MCPIP1 in adipogenesis. Here, we present evidence that Mcpip1 transcription is transiently activated during 3T3-L1 transition from pre- to adipocytes. However Mcpip1 protein expression is also strongly decreased at day one after induction of adipogenesis. Knockdown of Mcpip1 results in an upregulation of C/EBPß and PPARγ mRNAs, whereas overexpression of MCPIP1 reduces the level of both transcription factors and impairs adipogenesis. MCPIP1-dependend modulation of C/EBPß and PPARγ levels results in a modulation of the expression of downstream controlled genes. In addition, decreased C/EBPß, but not PPARγ, depends on the activity of the MCPIP1 PIN domain, which is responsible for RNase properties of this protein. Together, these data confirm that MCPIP1 is a key regulator of adipogenesis.

Targeting GD2 ganglioside and aurora A kinase as a dual strategy leading to cell death in cultures of human neuroblastoma cells.

The mechanism of the inhibitory effect of anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) on human neuroblastoma cells survival was studied in vitro. It was recently shown in IMR-32 cells that death induced by this antibody exhibited several characteristics typical of apoptosis. In this study we used cytotoxixity assays, qRT-PCR and immunoblotting to evaluate the response of several human neuroblastoma cell lines to the anti-GD2 14G2a mAb. We showed that the mAb decreases all three aurora kinases expression and phosphorylation in IMR-32 and LA-N-1 cells. Most importantly, we show, that MK-5108 specific aurora A kinase inhibitor decreases neuroblastoma cell survival, and when used in combination with the mAb, significantly potentiates cytotoxicity against IMR-32, CHP-134, and LA-N-5 neuroblastoma cells in vitro. It was shown that downregulation of aurora A kinase by the therapeutic antibody is associated with decreased levels of MYCN protein in cytoplasm, and induced expression of PHLDA1 and P53 proteins.

Prenatal exposure to antiandrogen flutamide affects androgen receptor (AR) expression in postnatal ovarian development in pig.

The following study was undertaken to localize androgen receptors (ARs) in various structures of the porcine ovary after prenatal exposure to antiandrogen flutamide. In utero treatment by antiandrogens may have adverse effects on reproductive function in immature and adult animals. Flutamide was injected into pregnant swines between days 20 and 28 (GD20) or 80 to 88 (GD80) of gestation. The ovaries were collected from treated animals and from control ones (non-treated) at two different points of development: from immature and adult pigs. Immunoexpression of AR was determined for preantral and antral follicles and for stroma cells. Immunostaining showed that AR expression in immature animals was unaffected in the primary follicles, while in the preantral and antral follicles the AR level fluctuated depending on day of treatment as well as on analyzed tissue. In adult animals, the immunoexpression of AR slightly decreased in antral follicles independently on the day of flutamide treatment. Therefore, AR expression in postnatal life may be affected by in utero exposure to antiandrogen flutamide.

Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1beta mRNA.

In human monocyte-derived macrophages, the MCPIP gene (monocyte chemoattractant protein-induced protein) is strongly activated by interleukin-1beta (IL-1beta). Using bioinformatics, a PIN domain was identified, spanning amino acids 130-280; such domains are known to possess structural features of RNases. Recently, RNase properties of MCPIP were confirmed on transcripts coding for interleukins IL-6 and IL-12p40. Here we present evidence that siRNA-mediated inhibition of the MCPIP gene expression increases the level of the IL-1beta transcript in cells stimulated with LPS, whereas overexpression of MCPIP exerts opposite effects. Cells with an increased level of wild-type MCPIP showed lower levels of IL-1beta mRNA. However, this was not observed when mutant forms of MCPIP, either entirely lacking the PIN domain or with point mutations in this domain, were used. The results of experiments with actinomycin D indicate that lower levels of IL-1beta mRNA are due to shortening of the IL-1beta transcript half-life, and are not related to the presence of AU-rich elements in the 3' UTR. The interaction of the MCPIP with transcripts of both IL-1beta and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes.

Mimitin - a novel cytokine-regulated mitochondrial protein.

BACKGROUND: The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners. RESULTS: By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway. CONCLUSION: Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.

Identification of interleukin-1 and interleukin-6-responsive genes in human monocyte-derived macrophages using microarrays.

The transcriptome profile of human monocyte-derived macrophages stimulated in vitro by low doses of IL-1 or IL-6 was analyzed by microarrays (Affymetrix, HG-U133A) in 5 independent experiments. Out of 4886 probe sets consistently detected in all 5 array replicates we found approximately 300 genes (FDR<5%) modulated by IL-1 and/or IL-6, among which 34 may be regarded as novel cytokine-responsive macrophage genes of various function. Detailed analysis indicates that cytokine-responsive genes include 125 transcripts significantly up-regulated by IL-1 and only 39 transcripts up-regulated by IL-6, whereas the number of down-regulated transcripts is lower and almost equal for both cytokines. These data indicate that, in comparison to liver cells, IL-1 is more potent than IL-6 in modulating gene expression of human macrophages. Hierarchical clustering analysis of these transcripts yielded 7 separate gene clusters. The most abundant group contains genes strongly activated by IL-1 alone and coding for chemokines, cytokines and their receptors, the components of intracellular signaling as well as transcription factors from NF-kB family. In order to validate the results obtained by microarray analysis the expression of 5 genes from various clusters was determined by quantitative RT-PCR. Moreover, the putative promoter regions of all cytokine-responsive genes were subjected to the in silico identification of transcription factor binding sites (TFBS). We found that TFBS corresponding to RelA/NF-kB is the most strongly over-represented group and we demonstrated involvement of NF-kB in the expression of selected genes.

Regulatory mechanisms of gene expression: complexity with elements of deterministic chaos.

Linear models based on proportionality between variables have been commonly applied in biology and medicine but in many cases they do not describe correctly the complex relationships of living organisms and now are being replaced by nonlinear theories of deterministic chaos. Recent advances in molecular biology and genome sequencing may lead to a simplistic view that all life processes in a cell, or in the whole organism, are strictly and in a linear fashion controlled by genes. In reality, the existing phenotype arises from a complex interaction of the genome and various environmental factors. Regulation of gene expression in the animal organism occurs at the level of epigenetic DNA modification, RNA transcription, mRNA translation, and many additional alterations of nascent proteins. The process of transcription is highly complicated and includes hundreds of transcription factors, enhancers and silencers, as well as various species of low molecular mass RNAs. In addition, alternative splicing or mRNA editing can generate a family of polypeptides from a single gene. Rearrangement of coding DNA sequences during somatic recombination is the source of great variability in the structure of immunoglobulins and some other proteins. The process of rearrangement of immunoglobulin genes, or such phenomena as parental imprinting of some genes, appear to occur in a random fashion. Therefore, it seems that the mechanism of genetic information flow from DNA to mature proteins does not fit the category of linear relationship based on simple reductionism or hard determinism but would be probably better described by nonlinear models, such as deterministic chaos.

A search for genes modulated by interleukin-6 alone or with interleukin-1beta in HepG2 cells using differential display analysis.

Interleukin-1 and interleukin-6 are principal cytokines involved in regulation of expression of acute-phase proteins. In the joint action of both cytokines IL-1 can suppress or enhance the IL-6-dependent induction of gene expression. Here, we report changes in the transcriptome profile of HepG2 cells exposed to IL-6 alone, or IL-1 and IL-6. Cytokine-responsive genes were identified by differential display analysis. Validation of observed changes in the transcript level was carried out using the slot blot method. Out of 88 cDNA species modulated by IL-6, only 38 represent different known genes whereas 18 clones match genomic clones in NCBI data with hypothetical cDNA sequences (the remaining 32 clones showed no homology with the database or represented several clones of the same gene). In the experiments with HepG2 cells prestimulated for 3 h with IL-1 and then stimulated with IL-6, 43 cDNA fragments were amplified. Twenty-three of them represent known genes while 10 clones have inserts matching hypothetical cDNA sequences in NCBI data. The identified transcripts modulated by IL-6 or both cytokines in HepG2 cells code for intracellular proteins of various function. The largest groups represent genes engaged in metabolism, protein synthesis and signaling pathways. Among all genes identified as differentially regulated under stimulation by IL-6, or IL-1/IL-6, six were detected in both types of stimulation. None of the typical genes coding for plasma acute phase proteins was identified in our experiments. This indicates that differential display cannot be used to characterize the profile of a given transcriptome. On the other hand, it is a useful technique for detection of new genes responding to IL-6 alone or IL-6 in combination with IL-1.

Cloning and characterization of 5' upstream promoter region of rat WAP gene.

Regulatory regions of genes encoding milk proteins are frequently used to produce in the mammary gland of transgenic animals a variety of pharmaceutically and medically important human proteins. One such example is the whey acidic protein (WAP) promoter region, identified so far in the genome of mouse, rat, rabbit, camel, pig, brushtail possum and Tammar wallaby. The aim of the present study was cloning and characterization of the 5' upstream promoter region of rat WAP gene. Using Genome Walking procedure, we cloned the region extending from -849 to -3671 bp. We have shown that there are two conserved regions highly similar to hypersensitive sites present in mouse and rabbit upstream region of WAP gene with binding sites for STAT5 transcription factor, essential for expression of WAP gene in mammary glands during lactation. We characterized dispersed and tandem repeats in the upstream region of rat WAP gen localized not far away from the translation initiation site.

Identification of changes in the transcriptome profile of human hepatoma HepG2 cells stimulated with interleukin-1 beta.

Interleukin-1 (IL-1) is the principal pro-inflammatory cytokine participating in the initiation of acute phase response. Human hepatoma HepG2 cells were exposed to 15 ng/ml of IL-1beta for times ranging from 1 to 24 h and the total RNA was isolated. Then cDNA was obtained and used for differential display with 10 arbitrary primers and 9 oligo(dT) primers designed by Clontech. Validation of observed changes of differentially expressed known genes was carried out by RT-PCR or Northern blot analysis. Out of 90 cDNA strands modulated by IL-1, 46 have been successfully reamplified and their sequencing indicates that they represent 36 different cDNA templates. By GenBank search, 26 cDNA clones were identified as already known genes while 10 showed no homology to any known gene. The identified transcripts modulated by IL-1 in HepG2 cells code for intracellular proteins of various function: trafficking/motor proteins (3 genes), proteins participating in the translation machinery or posttranscriptional/posttranslational modifications (7 genes), proteases (1 gene), proteins involved in metabolism (6 genes), activity modulators (3 genes), proteins of the cell cycle machinery (2 genes) and those functionally unclassified (4 genes). Majority of genes responded to IL-1 within 1 to 6 h (early genes), while two were late response genes (12-24 h) and four showed prolonged response over the whole 24-h period. Most of the observed changes of expression were in the range of two- to threefold increase in comparison to control untreated cells. Among identified genes, no typical secretory acute phase protein was found. The obtained results suggest that IL-1 affects the expression of several genes in HepG2 cells, especially those engaged in the synthesis and modifications of proteins.