Alexey Matskevich - Academia.edu (original) (raw)

Papers by Alexey Matskevich

Research paper thumbnail of Stimuli-dependent cleavage of Dicer during apoptosis

Biochemical Journal, Jul 1, 2008

miRNAs (microRNAs) play important roles in diverse physiological processes, including stress resp... more miRNAs (microRNAs) play important roles in diverse physiological processes, including stress response, apoptosis and carcinogenesis. Even though the role of individual miRNAs has been demonstrated, expression of proteins involved in miRNA production in response to acute stress or harmful agents has not been extensively investigated. Here, we have studied the role of Dicer, one of the central proteins of the miRNA processing machinery during apoptosis, and show that down-regulation of Dicer results in accelerated apoptosis of HeLa cells, triggered by TNFalpha (tumour necrosis factor alpha). We have also investigated the integrity of Dicer, and provide evidence that Dicer is a target for caspases during apoptosis. The cleavage of Dicer is stimulidependent and more pronounced when apoptosis is induced by PKC (protein kinase C) inhibitors, and can also be observed in HIV-1-infected cells at late stages of infection. Thus the apoptotic machinery may regulate the miRNA pathway by affecting individual proteins, such as Dicer.

Research paper thumbnail of Silencing of HIV by hairpin-loop-structured DNA oligonucleotide

Febs Letters, Jun 12, 2006

We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleoti... more We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple-helix formation. Upon treatment of HIV-infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN-mediated effect was highly sequence-specific. The ODN against HIV-IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV-BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.

Research paper thumbnail of Silencing of HIV RNA by a Hairpin-loop DNA

Retrovirology, 2005

We describe an RNA silencing, which inhibits HIV replication by a hairpin-loop DNA. A partially d... more We describe an RNA silencing, which inhibits HIV replication by a hairpin-loop DNA. A partially double-stranded 54mer DNA oligonucleotide (ODN) was targeted to the polypurine tract, PPT of HIV. It inhibits virus replication. We demonstrate that it prevents steps before DNA provirus formation. The effect of the ODN on HIV replication in cell culture is highly sequence-specific and sensitive to changes in length and single mismatches on either strand of the DNA. An ODN against HIV-IIIB was ineffective against HIV-Ba-L and vice versa, whereby their PPT's differ by two of 24 nucleotides. Thus, the structure and sequence of both strands of the ODN are important in cellular assays. In vitro the ODN leads to an RNA-DNA hybrid formation at the PPT, a structure which is cleaved by the RT/ RNase H in permeabilized virus particles. The hybrid at the PPT is preferentially recognized by the RT/RNase H for initiation of the second-strand DNA synthesis. This recognition is its normal biological function and shown here with the ODN. A cell extract containing cellular RNase H activities or RISC proteins, is unable to induce such a cleavage. In summary, the ODN mimicks a real step in viral replication, whereby the viral RNA is cleaved prematurely before DNA transcription is completed. The mechanism is reminiscent of RNA silencing by siRNA and supported by its relationship with RNaseH. The ODN may be a basis for drug design, because of low tendency for escape mutations. from 2005 International Meeting of The Institute of Human Virology

Research paper thumbnail of Silencing of viral RNAs by small double-stranded siDNA

Research paper thumbnail of Vero Cells as a Model to Study the Effects of Adenoviral Gene Delivery Vectors on the RNAi System in Context of Viral Infection

Journal of Innate Immunity, 2009

Technology based on RNA interference (RNAi) is a promising source for new antiviral therapies. Al... more Technology based on RNA interference (RNAi) is a promising source for new antiviral therapies. Although the application of RNAi has been studied extensively, significant problems with using RNAi remain. Very few studies have specifically assessed model systems for testing the effects of viruses or gene delivery vectors on the RNAi system. Since viruses have developed efficient strategies to circumvent the interferon (IFN) response, an IFN-deficient model system should be considered. Here we show that in Vero cells, which lack IFN-alpha and IFN-beta genes, knockdown of Dicer, a key RNAi component, led to accelerated death of cells infected with other evolutionary distinct viruses: influenza A virus, vesicular stomatitis virus and poliovirus. We also demonstrate that transduction of Vero cells with adenoviral vector with subsequent infection with influenza A virus also resulted in increased mortality of infected cells. These effects were much weaker in IFN-producing A549 and Hela cell lines. Thus, the Vero cell line could serve as an interesting model for studying the effects of gene delivery vectors on the RNAi system in the context of virus-related disorders.

Research paper thumbnail of Dicer is involved in protection against influenza A virus infection

Journal of General Virology, 2007

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the... more In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-a and IFN-b genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

Research paper thumbnail of 118 Self-inactivation of HIV by its own RT/RNase H

JAIDS Journal of Acquired Immune Deficiency Syndromes, 2009

Research paper thumbnail of Silencing of HIV by hairpin-loop-structured DNA oligonucleotide

FEBS Letters, 2006

We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleoti... more We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple-helix formation. Upon treatment of HIV-infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN-mediated effect was highly sequence-specific. The ODN against HIV-IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV-BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.

Research paper thumbnail of Chaperone activity of α-crystallin in relation to the sarcoplasmic reticulum Ca2+ pump of skeletal muscles in stress and adaptation

Bulletin of Experimental Biology and Medicine, 1999

a-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protect... more a-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protective effects on membrane systems of Ca 2. transport into the sarcoplasmic reticulum of skeletal muscles. Protective action of c~-crystallin depended on the body state. This effect was not observed in the control and after adaptation to stress, while after stress, especially against the background of adaptation, c~-crystallin increased the rate of Ca 2+ transport into the sarcoplasmic reticulum and thermal resistance of Ca 2+ pump. The mechanisms of c~-crystallin activation during stress are discussed.

Research paper thumbnail of The role of cytoplasmic factors in stabilization of Ca2+-transporting function of myocardial sarcoplasmic reticulum in rats during adaptation to stress

Bulletin of Experimental Biology and Medicine, 1999

Acute stress reduces and adaptation to stress enhances thermal resistance of Ca 2+ pump of the sa... more Acute stress reduces and adaptation to stress enhances thermal resistance of Ca 2+ pump of the sarcoplasmic reticulum fraction. Soluble cytoplasmic factors increase the rate of Ca 2 § transport into myocardial sarcoplasmic reticulum and its thermal resistance in the stressed, stress-adapted, and control rats, the activating effect being most pronounced during acute stress. Structural and functional mechanisms underlying the protective effect of soluble cytoplasmic factors on membrane-bound enzymes are discussed.

Research paper thumbnail of Stimuli-dependent cleavage of Dicer during apoptosis

Biochemical Journal, 2008

miRNAs (microRNAs) play important roles in diverse physiological processes, including stress resp... more miRNAs (microRNAs) play important roles in diverse physiological processes, including stress response, apoptosis and carcinogenesis. Even though the role of individual miRNAs has been demonstrated, expression of proteins involved in miRNA production in response to acute stress or harmful agents has not been extensively investigated. Here, we have studied the role of Dicer, one of the central proteins of the miRNA processing machinery during apoptosis, and show that down-regulation of Dicer results in accelerated apoptosis of HeLa cells, triggered by TNFalpha (tumour necrosis factor alpha). We have also investigated the integrity of Dicer, and provide evidence that Dicer is a target for caspases during apoptosis. The cleavage of Dicer is stimulidependent and more pronounced when apoptosis is induced by PKC (protein kinase C) inhibitors, and can also be observed in HIV-1-infected cells at late stages of infection. Thus the apoptotic machinery may regulate the miRNA pathway by affecting individual proteins, such as Dicer.

Research paper thumbnail of From TGN1412 to TAB08: the return of CD28 superagonist therapy to clinical development for the treatment of rheumatoid arthritis

Clinical and experimental rheumatology

CD28 superagonists (CD28SA) are CD28-specific monoclonal antibodies which are able to activate T-... more CD28 superagonists (CD28SA) are CD28-specific monoclonal antibodies which are able to activate T-cells without overt TCR engagement. In rodents, CD28SA efficiently activate regulatory T-cells and are therapeutically effective in multiple models of autoimmunity, inflammation and transplantation. However, a phase I study of the human CD28SA TGN1412 in 2006 resulted in a life-threatening cytokine storm. This brief review summarises preclinical work before and since the failed phase I trial with an emphasis on understanding the reasons why there had been no warning of toxicity, and how a novel assay paved the way for a new phase I, phase Ib (both completed), and an ongoing phase II study.

Research paper thumbnail of Exploiting hepatitis C virus activation of NFκB to deliver HCV-responsive expression of interferons α and γ

Gene Therapy, 2003

Chronic infection with hepatitis C virus (HCV) may lead to liver failure and hepatocellular carci... more Chronic infection with hepatitis C virus (HCV) may lead to liver failure and hepatocellular carcinoma. Current treatment for HCV includes high systemic doses of interferonα (IFNα), which is effective in less than half of patients and may have severe side effects. We designed conditional IFNα and IFNγ expression constructs to be triggered by HCV-induced activation of NFκB, and delivered these

Research paper thumbnail of Conditional Expression of IFN- and IFN- Activated by HBV as Genetic Therapy for Hepatitis B

J Interferon Cytokine Res, 2003

Research paper thumbnail of Short Partially Double-Stranded Oligodeoxynucleotide Induces Reverse Transcriptase/RNase H-Mediated Cleavage of HIV RNA and Contributes to Abrogation of Infectivity of Virions

Aids Research and Human Retroviruses, 2006

We describe a novel mechanism of viral RNA eradication by an oligodeoxynucleotide A (ODN A) direc... more We describe a novel mechanism of viral RNA eradication by an oligodeoxynucleotide A (ODN A) directly in HIV virions. The ODN A consists of an antisense and a passenger strand, and was designed to target the polyp-urine tract (PPT) of HIV-1, a conserved region of the viral genome. It leads to HIV reverse transcriptase/ribonuclease H (RT/RNase H)-dependent degradation of the RNA in viral particles. Illimaquinone, a specific inhibitor of RNase H, activity of HIV RT/RNase H, prevents RNA cleavage. The effect of the ODN A is sequence-specific and the passenger strand is important, since a lack or alteration of this strand reduces the antiviral activity of the ODN. ODN A has a stronger antiviral effect compared to a control ODN CO, targeted to a site outside of the PPT. The pretreatment with ODN A strongly reduced the infectivity of virions in cell culture in the absence of any DNA carriers or detergents.

Research paper thumbnail of Conditional expression of IFN-α and IFN-γ activated by HBV as genetic therapy for hepatitis B

Journal of Interferon Cytokine Research, 2003

Chronic infection with hepatitis B virus (HBV) has potentially devastating consequences and is ve... more Chronic infection with hepatitis B virus (HBV) has potentially devastating consequences and is very difficult to treat. Therapy with recombinant interferons (IFN), especially IFN-alpha, may be effective. The blood IFN-alpha levels that are needed to maintain therapeutic IFN-alpha levels in the liver, however, often cause severe side effects. Gene delivery to the liver may provide a solution. Using a long-term expression construct could provide the desired levels of IFN locally without the need to maintain potentially problematic blood levels. Recombinant, Tag-deleted SV40-derived vectors transduce hepatocytes efficiently and provide permanent transgene expression. We designed an expression construct that was effective against HBV and whose activity was limited to HBV-infected cells. To do this, we exploited the ability of HBV X protein to activate NF-kappaB and, via NF-kappaB, to activate promoter activity of HIV long terminal repeat (LTR) in hepatocytes. Using HIVLTR as a conditional promoter upstream of human and murine IFN-alpha and IFN-gamma cDNAs, rSV40 vectors were used to test the responsiveness of IFN to HBV and the ability of these IFNs to inhibit HBV transcripts and protein production and to activate IFN signaling in neighboring untransduced cells. We found that in hepatocyte cell lines and in primary hepatocytes, HBV activated the promoter activity of the HIVLTR via NF-kappaB. When whole HBV genome was delivered to cells by transfection to simulate HBV infection, IFN expression was activated, IFNs were produced and secreted, and they protected cells from HBV. Levels of IFN proteins that were secreted in this context were comparable to targeted blood levels needed to control chronic hepatitis viral infection. Further, IFNs that were elicited and secreted in this manner were able to activate IFN-induced signaling pathways in neighboring, untransduced cells and so were likely to provide protection even to cells that the rSV40 vector did not transduce. Gene delivery using such rSV40 vectors expressing IFNs conditionally in response to HBV may be an attractive therapeutic option for the treatment of chronic hepatitis B.

Research paper thumbnail of Dual Activation of the Drosophila Toll Pathway by TwoPattern Recognition Receptors

Research paper thumbnail of High-density preculture of PBMC restores defective sensitivity of circulating CD8 T-cells to virus- and tumor-derived antigens

Blood, Jan 29, 2015

Peripheral blood mononuclear cells (PBMC) are the only source of human lymphoid cells routinely a... more Peripheral blood mononuclear cells (PBMC) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T-cells transiently lose antigen sensitivity when cellular contacts are lost, e.g. by entering the circulation. Using the simple and robust RESTORE protocol for RESetting T-cells to Original Reactivity, i.e. pre-culturing PBMC for two days at a high cell density before initiation of antigenic stimulation, we here show that CD8 T-cell responses to viral and to tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of WT-1 specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of...

Research paper thumbnail of Paradigms for Conditional Expression of RNA Interference Molecules for Use Against Viral Targets

Methods in Enzymology, 2005

The rapid increase in the study of small interfering RNA (siRNA) as a means to decrease expressio... more The rapid increase in the study of small interfering RNA (siRNA) as a means to decrease expression of targeted genes has led to concerns about possible unexpected consequences of constitutive siRNA expression. We therefore devised a conditional siRNA expression system in which siRNA targeting hepatitis C virus (HCV) would be produced in response to HCV. We found that HCV acts via NFkappaB to stimulate the HIV long terminal repeat (LTR) as a promoter. We exploited this observation by designing conditional siRNA transcription constructs to be triggered by HCV-induced activation of NFkappaB. These were delivered by using highly efficient recombinant Tag-deleted SV40-derived vectors. Conditional activation of HIV-LTR and consequent siRNA synthesis in cells expressing HCV were observed. HCV-specific RNAi decreased HCV RNA greatly within 4 days, using transient transfection of the whole HCV genome as a model of acute HCV entry into transduced cells. We then tested the effectiveness of rSV40-delivered anti-HCV siRNA in cells stably transfected with the whole HCV genome to simulate hepatocytes chronically infected with HCV. There is considerable need for regulated production of siRNAs activated by a particular set of conditions (HCV in this case) but quiescent otherwise. Approaches described here may serve as a paradigm for such conditional siRNA expression.

Research paper thumbnail of Calpains: physiological and pathophysiological significance

Pathophysiology, 1999

The system of Ca-dependent neutral proteases, commonly known as the calpain system, is represente... more The system of Ca-dependent neutral proteases, commonly known as the calpain system, is represented by two main isoforms, calpain 1 and calpain 2, as well as by tissue-specific enzymes. These enzymes take part in some physiological processes and are responsible for certain pathological states. The versatility of intracellular Ca 2 + mediating the majority of cellular processes is reflected on the calpain system, i.e. it activates the functional reserves of the cell at moderate Ca 2 + concentrations and causes protein degradation at excessive Ca 2 + . Besides, the activity of the calpain system of the cell is regulated by the concentration of its specific inhibitor, calpastatin, as well as by phosphorylation-dephosphorylation of proteins. Calpains can function both inside the cell and outside it (in the extracellular matrix) and exist both in soluble and membrane-bound states, where it is the most active. Calpains induce cell degradation in some pathologies and in apoptosis. Preliminary modification of proteins or their membrane microenvironment initiates enhanced proteolysis of cytosolic and membrane-bound proteins and enzymes. The key role of calpains in the development of many pathological states testifies to the high pathophysiological significance of this proteolytic system in the organism.

Research paper thumbnail of Stimuli-dependent cleavage of Dicer during apoptosis

Biochemical Journal, Jul 1, 2008

miRNAs (microRNAs) play important roles in diverse physiological processes, including stress resp... more miRNAs (microRNAs) play important roles in diverse physiological processes, including stress response, apoptosis and carcinogenesis. Even though the role of individual miRNAs has been demonstrated, expression of proteins involved in miRNA production in response to acute stress or harmful agents has not been extensively investigated. Here, we have studied the role of Dicer, one of the central proteins of the miRNA processing machinery during apoptosis, and show that down-regulation of Dicer results in accelerated apoptosis of HeLa cells, triggered by TNFalpha (tumour necrosis factor alpha). We have also investigated the integrity of Dicer, and provide evidence that Dicer is a target for caspases during apoptosis. The cleavage of Dicer is stimulidependent and more pronounced when apoptosis is induced by PKC (protein kinase C) inhibitors, and can also be observed in HIV-1-infected cells at late stages of infection. Thus the apoptotic machinery may regulate the miRNA pathway by affecting individual proteins, such as Dicer.

Research paper thumbnail of Silencing of HIV by hairpin-loop-structured DNA oligonucleotide

Febs Letters, Jun 12, 2006

We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleoti... more We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple-helix formation. Upon treatment of HIV-infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN-mediated effect was highly sequence-specific. The ODN against HIV-IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV-BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.

Research paper thumbnail of Silencing of HIV RNA by a Hairpin-loop DNA

Retrovirology, 2005

We describe an RNA silencing, which inhibits HIV replication by a hairpin-loop DNA. A partially d... more We describe an RNA silencing, which inhibits HIV replication by a hairpin-loop DNA. A partially double-stranded 54mer DNA oligonucleotide (ODN) was targeted to the polypurine tract, PPT of HIV. It inhibits virus replication. We demonstrate that it prevents steps before DNA provirus formation. The effect of the ODN on HIV replication in cell culture is highly sequence-specific and sensitive to changes in length and single mismatches on either strand of the DNA. An ODN against HIV-IIIB was ineffective against HIV-Ba-L and vice versa, whereby their PPT's differ by two of 24 nucleotides. Thus, the structure and sequence of both strands of the ODN are important in cellular assays. In vitro the ODN leads to an RNA-DNA hybrid formation at the PPT, a structure which is cleaved by the RT/ RNase H in permeabilized virus particles. The hybrid at the PPT is preferentially recognized by the RT/RNase H for initiation of the second-strand DNA synthesis. This recognition is its normal biological function and shown here with the ODN. A cell extract containing cellular RNase H activities or RISC proteins, is unable to induce such a cleavage. In summary, the ODN mimicks a real step in viral replication, whereby the viral RNA is cleaved prematurely before DNA transcription is completed. The mechanism is reminiscent of RNA silencing by siRNA and supported by its relationship with RNaseH. The ODN may be a basis for drug design, because of low tendency for escape mutations. from 2005 International Meeting of The Institute of Human Virology

Research paper thumbnail of Silencing of viral RNAs by small double-stranded siDNA

Research paper thumbnail of Vero Cells as a Model to Study the Effects of Adenoviral Gene Delivery Vectors on the RNAi System in Context of Viral Infection

Journal of Innate Immunity, 2009

Technology based on RNA interference (RNAi) is a promising source for new antiviral therapies. Al... more Technology based on RNA interference (RNAi) is a promising source for new antiviral therapies. Although the application of RNAi has been studied extensively, significant problems with using RNAi remain. Very few studies have specifically assessed model systems for testing the effects of viruses or gene delivery vectors on the RNAi system. Since viruses have developed efficient strategies to circumvent the interferon (IFN) response, an IFN-deficient model system should be considered. Here we show that in Vero cells, which lack IFN-alpha and IFN-beta genes, knockdown of Dicer, a key RNAi component, led to accelerated death of cells infected with other evolutionary distinct viruses: influenza A virus, vesicular stomatitis virus and poliovirus. We also demonstrate that transduction of Vero cells with adenoviral vector with subsequent infection with influenza A virus also resulted in increased mortality of infected cells. These effects were much weaker in IFN-producing A549 and Hela cell lines. Thus, the Vero cell line could serve as an interesting model for studying the effects of gene delivery vectors on the RNAi system in the context of virus-related disorders.

Research paper thumbnail of Dicer is involved in protection against influenza A virus infection

Journal of General Virology, 2007

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the... more In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-a and IFN-b genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

Research paper thumbnail of 118 Self-inactivation of HIV by its own RT/RNase H

JAIDS Journal of Acquired Immune Deficiency Syndromes, 2009

Research paper thumbnail of Silencing of HIV by hairpin-loop-structured DNA oligonucleotide

FEBS Letters, 2006

We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleoti... more We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple-helix formation. Upon treatment of HIV-infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN-mediated effect was highly sequence-specific. The ODN against HIV-IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV-BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.

Research paper thumbnail of Chaperone activity of α-crystallin in relation to the sarcoplasmic reticulum Ca2+ pump of skeletal muscles in stress and adaptation

Bulletin of Experimental Biology and Medicine, 1999

a-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protect... more a-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protective effects on membrane systems of Ca 2. transport into the sarcoplasmic reticulum of skeletal muscles. Protective action of c~-crystallin depended on the body state. This effect was not observed in the control and after adaptation to stress, while after stress, especially against the background of adaptation, c~-crystallin increased the rate of Ca 2+ transport into the sarcoplasmic reticulum and thermal resistance of Ca 2+ pump. The mechanisms of c~-crystallin activation during stress are discussed.

Research paper thumbnail of The role of cytoplasmic factors in stabilization of Ca2+-transporting function of myocardial sarcoplasmic reticulum in rats during adaptation to stress

Bulletin of Experimental Biology and Medicine, 1999

Acute stress reduces and adaptation to stress enhances thermal resistance of Ca 2+ pump of the sa... more Acute stress reduces and adaptation to stress enhances thermal resistance of Ca 2+ pump of the sarcoplasmic reticulum fraction. Soluble cytoplasmic factors increase the rate of Ca 2 § transport into myocardial sarcoplasmic reticulum and its thermal resistance in the stressed, stress-adapted, and control rats, the activating effect being most pronounced during acute stress. Structural and functional mechanisms underlying the protective effect of soluble cytoplasmic factors on membrane-bound enzymes are discussed.

Research paper thumbnail of Stimuli-dependent cleavage of Dicer during apoptosis

Biochemical Journal, 2008

miRNAs (microRNAs) play important roles in diverse physiological processes, including stress resp... more miRNAs (microRNAs) play important roles in diverse physiological processes, including stress response, apoptosis and carcinogenesis. Even though the role of individual miRNAs has been demonstrated, expression of proteins involved in miRNA production in response to acute stress or harmful agents has not been extensively investigated. Here, we have studied the role of Dicer, one of the central proteins of the miRNA processing machinery during apoptosis, and show that down-regulation of Dicer results in accelerated apoptosis of HeLa cells, triggered by TNFalpha (tumour necrosis factor alpha). We have also investigated the integrity of Dicer, and provide evidence that Dicer is a target for caspases during apoptosis. The cleavage of Dicer is stimulidependent and more pronounced when apoptosis is induced by PKC (protein kinase C) inhibitors, and can also be observed in HIV-1-infected cells at late stages of infection. Thus the apoptotic machinery may regulate the miRNA pathway by affecting individual proteins, such as Dicer.

Research paper thumbnail of From TGN1412 to TAB08: the return of CD28 superagonist therapy to clinical development for the treatment of rheumatoid arthritis

Clinical and experimental rheumatology

CD28 superagonists (CD28SA) are CD28-specific monoclonal antibodies which are able to activate T-... more CD28 superagonists (CD28SA) are CD28-specific monoclonal antibodies which are able to activate T-cells without overt TCR engagement. In rodents, CD28SA efficiently activate regulatory T-cells and are therapeutically effective in multiple models of autoimmunity, inflammation and transplantation. However, a phase I study of the human CD28SA TGN1412 in 2006 resulted in a life-threatening cytokine storm. This brief review summarises preclinical work before and since the failed phase I trial with an emphasis on understanding the reasons why there had been no warning of toxicity, and how a novel assay paved the way for a new phase I, phase Ib (both completed), and an ongoing phase II study.

Research paper thumbnail of Exploiting hepatitis C virus activation of NFκB to deliver HCV-responsive expression of interferons α and γ

Gene Therapy, 2003

Chronic infection with hepatitis C virus (HCV) may lead to liver failure and hepatocellular carci... more Chronic infection with hepatitis C virus (HCV) may lead to liver failure and hepatocellular carcinoma. Current treatment for HCV includes high systemic doses of interferonα (IFNα), which is effective in less than half of patients and may have severe side effects. We designed conditional IFNα and IFNγ expression constructs to be triggered by HCV-induced activation of NFκB, and delivered these

Research paper thumbnail of Conditional Expression of IFN- and IFN- Activated by HBV as Genetic Therapy for Hepatitis B

J Interferon Cytokine Res, 2003

Research paper thumbnail of Short Partially Double-Stranded Oligodeoxynucleotide Induces Reverse Transcriptase/RNase H-Mediated Cleavage of HIV RNA and Contributes to Abrogation of Infectivity of Virions

Aids Research and Human Retroviruses, 2006

We describe a novel mechanism of viral RNA eradication by an oligodeoxynucleotide A (ODN A) direc... more We describe a novel mechanism of viral RNA eradication by an oligodeoxynucleotide A (ODN A) directly in HIV virions. The ODN A consists of an antisense and a passenger strand, and was designed to target the polyp-urine tract (PPT) of HIV-1, a conserved region of the viral genome. It leads to HIV reverse transcriptase/ribonuclease H (RT/RNase H)-dependent degradation of the RNA in viral particles. Illimaquinone, a specific inhibitor of RNase H, activity of HIV RT/RNase H, prevents RNA cleavage. The effect of the ODN A is sequence-specific and the passenger strand is important, since a lack or alteration of this strand reduces the antiviral activity of the ODN. ODN A has a stronger antiviral effect compared to a control ODN CO, targeted to a site outside of the PPT. The pretreatment with ODN A strongly reduced the infectivity of virions in cell culture in the absence of any DNA carriers or detergents.

Research paper thumbnail of Conditional expression of IFN-α and IFN-γ activated by HBV as genetic therapy for hepatitis B

Journal of Interferon Cytokine Research, 2003

Chronic infection with hepatitis B virus (HBV) has potentially devastating consequences and is ve... more Chronic infection with hepatitis B virus (HBV) has potentially devastating consequences and is very difficult to treat. Therapy with recombinant interferons (IFN), especially IFN-alpha, may be effective. The blood IFN-alpha levels that are needed to maintain therapeutic IFN-alpha levels in the liver, however, often cause severe side effects. Gene delivery to the liver may provide a solution. Using a long-term expression construct could provide the desired levels of IFN locally without the need to maintain potentially problematic blood levels. Recombinant, Tag-deleted SV40-derived vectors transduce hepatocytes efficiently and provide permanent transgene expression. We designed an expression construct that was effective against HBV and whose activity was limited to HBV-infected cells. To do this, we exploited the ability of HBV X protein to activate NF-kappaB and, via NF-kappaB, to activate promoter activity of HIV long terminal repeat (LTR) in hepatocytes. Using HIVLTR as a conditional promoter upstream of human and murine IFN-alpha and IFN-gamma cDNAs, rSV40 vectors were used to test the responsiveness of IFN to HBV and the ability of these IFNs to inhibit HBV transcripts and protein production and to activate IFN signaling in neighboring untransduced cells. We found that in hepatocyte cell lines and in primary hepatocytes, HBV activated the promoter activity of the HIVLTR via NF-kappaB. When whole HBV genome was delivered to cells by transfection to simulate HBV infection, IFN expression was activated, IFNs were produced and secreted, and they protected cells from HBV. Levels of IFN proteins that were secreted in this context were comparable to targeted blood levels needed to control chronic hepatitis viral infection. Further, IFNs that were elicited and secreted in this manner were able to activate IFN-induced signaling pathways in neighboring, untransduced cells and so were likely to provide protection even to cells that the rSV40 vector did not transduce. Gene delivery using such rSV40 vectors expressing IFNs conditionally in response to HBV may be an attractive therapeutic option for the treatment of chronic hepatitis B.

Research paper thumbnail of Dual Activation of the Drosophila Toll Pathway by TwoPattern Recognition Receptors

Research paper thumbnail of High-density preculture of PBMC restores defective sensitivity of circulating CD8 T-cells to virus- and tumor-derived antigens

Blood, Jan 29, 2015

Peripheral blood mononuclear cells (PBMC) are the only source of human lymphoid cells routinely a... more Peripheral blood mononuclear cells (PBMC) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T-cells transiently lose antigen sensitivity when cellular contacts are lost, e.g. by entering the circulation. Using the simple and robust RESTORE protocol for RESetting T-cells to Original Reactivity, i.e. pre-culturing PBMC for two days at a high cell density before initiation of antigenic stimulation, we here show that CD8 T-cell responses to viral and to tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of WT-1 specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of...

Research paper thumbnail of Paradigms for Conditional Expression of RNA Interference Molecules for Use Against Viral Targets

Methods in Enzymology, 2005

The rapid increase in the study of small interfering RNA (siRNA) as a means to decrease expressio... more The rapid increase in the study of small interfering RNA (siRNA) as a means to decrease expression of targeted genes has led to concerns about possible unexpected consequences of constitutive siRNA expression. We therefore devised a conditional siRNA expression system in which siRNA targeting hepatitis C virus (HCV) would be produced in response to HCV. We found that HCV acts via NFkappaB to stimulate the HIV long terminal repeat (LTR) as a promoter. We exploited this observation by designing conditional siRNA transcription constructs to be triggered by HCV-induced activation of NFkappaB. These were delivered by using highly efficient recombinant Tag-deleted SV40-derived vectors. Conditional activation of HIV-LTR and consequent siRNA synthesis in cells expressing HCV were observed. HCV-specific RNAi decreased HCV RNA greatly within 4 days, using transient transfection of the whole HCV genome as a model of acute HCV entry into transduced cells. We then tested the effectiveness of rSV40-delivered anti-HCV siRNA in cells stably transfected with the whole HCV genome to simulate hepatocytes chronically infected with HCV. There is considerable need for regulated production of siRNAs activated by a particular set of conditions (HCV in this case) but quiescent otherwise. Approaches described here may serve as a paradigm for such conditional siRNA expression.

Research paper thumbnail of Calpains: physiological and pathophysiological significance

Pathophysiology, 1999

The system of Ca-dependent neutral proteases, commonly known as the calpain system, is represente... more The system of Ca-dependent neutral proteases, commonly known as the calpain system, is represented by two main isoforms, calpain 1 and calpain 2, as well as by tissue-specific enzymes. These enzymes take part in some physiological processes and are responsible for certain pathological states. The versatility of intracellular Ca 2 + mediating the majority of cellular processes is reflected on the calpain system, i.e. it activates the functional reserves of the cell at moderate Ca 2 + concentrations and causes protein degradation at excessive Ca 2 + . Besides, the activity of the calpain system of the cell is regulated by the concentration of its specific inhibitor, calpastatin, as well as by phosphorylation-dephosphorylation of proteins. Calpains can function both inside the cell and outside it (in the extracellular matrix) and exist both in soluble and membrane-bound states, where it is the most active. Calpains induce cell degradation in some pathologies and in apoptosis. Preliminary modification of proteins or their membrane microenvironment initiates enhanced proteolysis of cytosolic and membrane-bound proteins and enzymes. The key role of calpains in the development of many pathological states testifies to the high pathophysiological significance of this proteolytic system in the organism.