Alf Grandien - Academia.edu (original) (raw)

Papers by Alf Grandien

Research paper thumbnail of Gene expression profiling of erythroblasts from refractory anaemia with ring sideroblasts (RARS) and effects of G-CSF

British Journal of Haematology, 2009

Research paper thumbnail of 5-Fluorouracil-induced RNA stress engages a TRAIL-DISC-dependent apoptosis axis facilitated by p53

Oncotarget, Jan 22, 2015

Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains... more Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-tr...

Research paper thumbnail of cIAP-2 block apoptotic events in bladder cancer cells

Anticancer research

The Inhibitors of Apoptosis (IAPs) are negative regulators of apoptosis and their overexpression ... more The Inhibitors of Apoptosis (IAPs) are negative regulators of apoptosis and their overexpression renders cells resistant to a variety of apoptotic stimuli. We investigated the mRNA expression levels of IAPs in a panel of bladder tumour cells, selected as being sensitive or resistant to death receptor-mediated apoptosis. The mRNA expression of IAPs was quantified in a RNase protection assay (RPA). Apoptosis was induced by recombinant killerTRAIL, agonistic anti-CD95 mAbs or doxorubicin and quantified by TUNEL and MTT assays. Stable expression of cIAP-2 was obtained by retroviral transduction. The expression of cIAP-2 mRNA was highly correlated with resistance to TRAIL-mediated apoptosis. Overexpression of cIAP-2 conferred resistance to previously sensitive cell lines and made cells less susceptible to doxorubicin. Treatment with doxorubicin in combination with TRAIL or anti-CD95 resulted in a synergistic cytotoxic effect, which was not possible to reverse by overexpression of cIAP-2....

Research paper thumbnail of High level of cFLIP correlates with resistance to death receptor-induced apoptosis in bladder carcinoma cells

Anticancer research

The cellular form of FLICE-inhibitory protein (cFLIP) blocks death receptor-induced apoptosis and... more The cellular form of FLICE-inhibitory protein (cFLIP) blocks death receptor-induced apoptosis and has been implicated in tumour progression. cFLIP interacts with caspase-8, thereby preventing activation of the caspase cascade. In this study we investigated the endogenous expression of cFLIP and caspase-8 in bladder carcinoma cells in relation to their sensitivity to death receptor-ligation. Apoptosis was induced by agonistic anti-CD95 mAbs or recombinant TRAIL and quantified by the TUNEL technique. The relative mRNA expression of cFLIP and caspase-8 was quantified by real-time PCR. Stable expression of cFLIP long (cFLIPL) was obtained by retroviral transduction. The relative ratio of cFLIP and caspase-8 was directly correlated to resistance to anti-CD95 or TRAIL-mediated apoptosis. Overexpression of cFLIPL shifted the responsiveness towards resistant status. cFLIP is an important determinant of susceptibility to death receptor-induced apoptosis in bladder carcinomas and could functi...

Research paper thumbnail of Multiparametric analysis of antibody repertoires

Research paper thumbnail of Regulated ChAT journal.pone.0065936

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells... more Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer's disease and multiple sclerosis in particular.

Research paper thumbnail of Development of B Cells secreting Endogenous or Transgene-Encoded Immunoglobulins in H-Chain Transgenic Mice

Scandinavian Journal of Immunology, 1993

The development of splenic B cells secreting transgene-encoded or endogenous immunoglobulin (Ig) ... more The development of splenic B cells secreting transgene-encoded or endogenous immunoglobulin (Ig) was analysed in the mu heavy (H-)chain transgenic mouse line M54. The results show that cells secreting endogenous Ig are not detectable during the perinatal period, even after lipopolysaccharide stimulation in vitro. At this time, transgene-secreting cells are readily detectable and keep increasing with age of the animals. After a few weeks of age cells secreting endogenous Ig appear in the spleen and keep increasing with age, reaching numbers comparable to non-transgenic littermates by 5 weeks of age. Thereafter, the proportion of transgene-secreting B cells decreases. We conclude that the preferential expression of endogenous Igs by secreting B cells in the adult does not result from peculiar genetic features of those cells, but from age-dependent cellular selection operating on all B cells.

Research paper thumbnail of Angiostatic Effects of NK Cell-Derived IFN- γ Counteracted by Tumour Cell Bcl-x L Expression

Scandinavian Journal of Immunology, 2014

Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the... more Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the antiapoptotic protein Bcl-x L , known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour-progressiveactivity in amurinelymphoma model. Bcl-x L overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl-x L protection of tumours from NK cellmediated attack did not involve protection from NK cell-mediated cytotoxicity. Instead, Bcl-x L -blocked apoptosis resulting from hypoxia and/or nutrient loss associated with theinhibition of angiogenesiscaused by NK cell-secreted IFN-c. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.

Research paper thumbnail of Simultaneous Expression and Detection of Multiple Retroviral Constructs in Haematopoietic Cells After Bone Marrow Transplantation

Scandinavian Journal of Immunology, 2005

Due to the complexity and redundancy of molecular processes governing the development and functio... more Due to the complexity and redundancy of molecular processes governing the development and function of haematopoietic cells, experimental procedures allowing simultaneous alteration in gene expression of multiple genes in vivo are needed. Here, we describe a protocol allowing for simultaneous transduction of haematopoietic stem cells (HSC) with two different replication incompetent retroviral expression vectors followed by transplantation of lethally irradiated recipient mice. These bicistronic retroviral vectors carried genes for the enhanced green and yellow florescent proteins (EGFP and EYFP) respectively. Spleen cells from reconstituted animals were stained for common lymphocyte and myeloid markers, then analysed on a two-laser, 488 and 635 nm, flow cytometer equipped with a 510/20-nm bandpass filter for FL1, a 550/30-nm bandpass filter for FL2 and a 530-nm short-pass dichroic mirror. It was demonstrated that cells expressing EGFP, EYFP or combinations thereof could be distinguished and analysed for staining with PerCP-and APC-conjugated reagents. We found that a sizable proportion of cells (70%) from reconstituted animals expressed EGFP and/or EYFP and that expression of these genes did not affect lymphoid or myeloid development. We also demonstrated that the alternative optical configuration allowed for conventional multiparameter flow cytometric analyses.

Research paper thumbnail of Participation of FLIP, RIP and Bcl-x L in Fas-mediated T-cell Death

Scandinavian Journal of Immunology, 2007

Apart from the conventional Fas signalling pathway, alternative pathways including the mitochondr... more Apart from the conventional Fas signalling pathway, alternative pathways including the mitochondrial caspase-dependent and RIP-mediated cell death routes have been proposed to operate during Fas-mediated cell death. To evaluate the contribution of different Fas signalling pathways, mice overexpressing FLIP(L), Bcl-x(L), a kinase-deficient form of RIP (RIPDeltakin) or combinations thereof were generated by retroviral gene transfer of haematopoietic stem cells. Such mice did not show overt abnormalities in haematopoietic development, defects in thymic deletion, accumulation of double-negative T cells or signs of autoimmunity. Fas-mediated death of mitogen-activated T cells was caspase dependent and could be blocked by FLIP(L) overexpression only with the minor involvement of Bcl-x(L) or RIPDeltakin inhibitable pathways. Fas-mediated death of resting CD4(+) and CD8(+) T cells was mainly caspase dependent but could only partly be blocked by FLIP(L) overexpression. Both Bcl-x(L) or RIPDeltakin expression resulted in partial protection of CD8(+) T cells against Fas-mediated cell death. These results indicate that yet uncharacterized signalling pathways from the Fas receptor are critically involved in lymphoproliferative and autoimmune disease observed in lpr mice and autoimmune lymphoproliferative syndrome patients.

Research paper thumbnail of Acquisition of CD40 Expression During Murine B-Cell Differentiation

Scandinavian Journal of Immunology, 1996

Expression of CD40 on mouse cells was investigated comparing the binding to cells of a monoclonal... more Expression of CD40 on mouse cells was investigated comparing the binding to cells of a monoclonal antibody against CD40, to that of a soluble fusion protein consisting of the extracellular domains of the mouse CD40-ligand. The analysis of a series of established cell lines failed to demonstrate expression of CD40 on pro- or pre-B cells, and indicated that CD40 expression was restricted to cells that had undergone productive heavy- and light-chain gene rearrangements, and expressed surface Ig. In cells from normal mice, CD40 first becomes detectable, although at low levels, on a subset of small pre-B-II cells in bone marrow, the levels of CD40 expression increasing thereafter during B-cell maturation. Thus, immature B cells (IgM+ IgDlo B220lo) express intermediate levels of CD40, and mature B cells (IgM+ IgDhi B220hi) express high levels of CD40. Anatomical location also seems to correlate with the levels of CD40 expression, as B cells expressing the highest levels of CD40 were found in lymph nodes and Peyer's patches.

Research paper thumbnail of Inhibition of the Intrinsic but Not the Extrinsic Apoptosis Pathway Accelerates and Drives Myc-Driven Tumorigenesis Towards Acute Myeloid Leukemia

PLoS ONE, 2012

Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However... more Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However, MYC is also a powerful inducer of apoptosis, which is one of the major failsafe programs to prevent cancer development. To clarify the relative importance of the extrinsic (death receptor-mediated) versus the intrinsic (mitochondrial) pathway of apoptosis in MYC-driven AML, we coexpressed MYC together with anti-apoptotic proteins of relevance for AML; BCL-X(L)/BCL-2 (inhibiting the intrinsic pathway) or FLIP(L) (inhibiting the extrinsic pathway), in hematopoietic stems cells (HSCs). Transplantation of HSCs expressing MYC into syngeneic recipient mice resulted in development of AML and T-cell lymphomas within 7-9 weeks as expected. Importantly, coexpression of MYC together with BCL-X(L)/BCL-2 resulted in strongly accelerated kinetics and favored tumor development towards aggressive AML. In contrast, coexpression of MYC and FLIP(L) did neither accelerate tumorigenesis nor change the ratio of AML versus T-cell lymphoma. However, a change in distribution of immature CD4(+)CD8(+) versus mature CD4(+) T-cell lymphoma was observed in MYC/FLIP(L) mice, possibly as a result of increased survival of the CD4+ population, but this did not significantly affect the outcome of the disease. In conclusion, our findings provide direct evidence that BCL-X(L) and BCL-2 but not FLIP(L) acts in synergy with MYC to drive AML development.

Research paper thumbnail of DISC-mediated activation of caspase-2 in DNA damage-induced apoptosis

Oncogene, 2009

The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signal... more The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signaling cascade regulated by sequential activation of caspases. Mechanisms that lead from p53 to activation of individual initiator caspases are still unclear. The present model for caspase-2 activation includes PIDDosome complex formation. However, in certain experimental models, elimination of complex constituents PIDD or RAIDD did not significantly influence caspase-2 activation, suggesting the existence of an alternative activation platform for caspase-2. Here we have investigated the link between p53 and caspase-2 in further detail and report that the latter is able to utilize the CD95 DISC as an activation platform. The recruitment of caspase-8 to this complex is required for activation of caspase-2. In the experimental system used, the DISC is formed through a distinct, p53-dependent upregulation of CD95. Moreover, we show that caspase-2 and -8 cleave Bid, and that both act simultaneously upstream of mitochondrial cytochrome c release. Finally, a direct interaction between the two caspases and the ability of caspase-8 to cleave caspase-2 are demonstrated. Thus, the observed functional link between caspase-8 and -2 within the DISC represents an alternative mechanism to the PIDDosome for caspase-2 activation in response to DNA damage.

Research paper thumbnail of Host strain-dependent difference in susceptibility in a rat model of herpes simplex type 1 encephalitis

Journal of Neurovirology, 2008

Herpes simplex encephalitis (HSE) is characterized by severe focal brain inflammation leading to ... more Herpes simplex encephalitis (HSE) is characterized by severe focal brain inflammation leading to substantial loss of nervous tissue. The authors established a model of Herpes simplex virus type 1 (HSV)-1-induced acute encephalitis in the rat by injecting into the whiskers' area a virus strain isolated from a fatal human HSE case. The model might resemble natural propagation of HSV-1 in humans; spreading from the mouth and lips via the trigeminal nerve to trigeminal ganglia and subsequently entering the central nervous system (CNS). HSV-1 infected Dark Agouti (DA) rats developed a well-synchronized disease and died 5 days after inoculation. HSV-1 detection by quantitative polymerase chain reaction (qPCR), virus isolation and immunohistochemistry, magnetic resonance imaging, and histopathological examination verified dramatic encephalitis mainly in the brainstem, but also in the olfactory bulb and other segments of the brain of diseased rats. In contrast, Piebald Virol Glaxo (PVG) rats were completely resistant to disease, displaying a more rapid clearance of peripheral infection and no evidence of virus entering into neither the trigeminal ganglia nor the CNS. These results suggest a regulation of susceptibility to HSV-1-induced encephalitis at the level of peripheral infection and subsequent neuronal uptake/transport of the virus. This provides a basis for future positioning of genetic polymorphisms regulating HSE and for dissection of important pathogenetic mechanisms of this severe human disease.

Research paper thumbnail of Expression of the Long Form of Human FLIP by Retroviral Gene Transfer of Hemopoietic Stem Cells Exacerbates Experimental Autoimmune Encephalomyelitis

The Journal of Immunology, 2003

To test this concept, we examined the effects of overexpressing the long form of human FLICEinhib... more To test this concept, we examined the effects of overexpressing the long form of human FLICEinhibitory protein, a potent inhibitor of death receptor-mediated apoptosis, in myelin oligodendrocyte glycoprotein-induced EAE in DBA/1 mice. We found that overexpression of the long form of human FLICE-inhibitory protein by retroviral gene transfer of hemopoietic stem cells led to a clinically more severe EAE in these mice compared with control mice receiving the retroviral vector alone. The exacerbated disease was evident by an enhanced and prolonged inflammatory reaction in the CNS of these animals compared with control mice.

Research paper thumbnail of Death Receptor Ligation or Exposure to Perforin Trigger Rapid Egress of the Intracellular Parasite Toxoplasma gondii

The Journal of Immunology, 2007

Research paper thumbnail of Expression and Selection of Murine Antibody Repertoires

International Reviews of Immunology, 1992

Research paper thumbnail of Endogenous V H gene family expression in immunoglobulin-transgenic mice: evidence for selection of antibody repertoires

International Immunology, 1991

VH gene family expression in single cells of the emergent, available and actual B cell repertoire... more VH gene family expression in single cells of the emergent, available and actual B cell repertoires of C57BL/6 mice was compared to that of two immunoglobulin (Ig)-transgenic B6 lines (B6-Sp6 and M54). We found that less than 5% of bone marrow cells of transgenic mice express endogenous VH genes and that the vast majority (95%) of the peripheral, mature B cell repertoire in these animals is composed of cells expressing the VHJ558 transgenic family. Unimmunized transgenic mice, however, diversify VH gene family usage by 'background' Ig-secreting cells in the spleen, greater than 50% of which express endogenous VH genes. The pattern of endogenous VH gene family expression in the actual repertoire of B6-Sp6 mice is indistinguishable from that of normal B6 mice. In contrast, actual repertoires of M54 mice differ by a 4- to 5-fold higher representation of the VHQ52 family. These results demonstrate a powerful positive selection of B cells into the secretory compartments of unimmunized animals, show that actual and available repertoires differ very markedly, and suggest that V region interactions participate in the selection of 'natural antibody' repertoires.

Research paper thumbnail of An example of idiotypic mimicry

European Journal of Immunology, 1997

We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibod... more We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibody repertoires. The transgenic system was chosen as to allow for normal recombination of endogenous Ig genes, secretion of TGIg from early development on, and distinguishing the TGIg from endogenous Ig by several serological markers on the C and V regions of the molecules. The transgenic construct encodes a complete anti-(4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NP) antibody molecule carrying a well-defined idiotype, bearing a lambda 1 light chain and a chimeric heavy chain encoded by a human alpha 2 C region devoid of its membrane exon, and the murine B1.8 VDJ-region. Endogenous antibody repertoires were analyzed in mitogen-driven limiting dilution cultures, in single-cell assays for naturally activated Ig-secreting cells, and in hybridomas derived by direct fusion of spleen cells from unmanipulated animals. The results show that a very high frequency of splenic resting B cells and plasma cells in transgenic animals produce IgM with B1.8-cross-reactive idiotypes. This was confirmed by hybridoma analysis which also established that the levels of transgene expression and of idiotype-positive IgM production by the same cell are not correlated. The affinities of idiotype-positive endogenous Ig varied, but were generally several orders of magnitude lower than the transgene-encoded idiotype. V regions from idiotype-cross-reactive IgM heavy chains showed marked diversity in sequences that were all different from the transgenic B1.8. These results are compatible with idiotypic mimicry resulting from intercellular selection based on degenerate, whole V region reactivities.

Research paper thumbnail of Suppression of B cell differentiation by ligation of membrane-bound IgM

European Journal of Immunology, 1993

Using B cells from the transgenic mouse line B6-Sp6 and control littermates, stimulated by lipopo... more Using B cells from the transgenic mouse line B6-Sp6 and control littermates, stimulated by lipopolysaccharide (LPS) under novel culture conditions that provide for the response of all B cells, we show here that specific ligation of the surface IgM molecules always results in inhibition of terminal differentiation and immunoglobulin secretion by activated cells, regardless of the ligand. Thus, monoclonal antibodies to (a) the CH region of Ig (anti-mu and anti-allotype), (b) the C kappa region, (c) the V region (anti-idiotype) of surface IgM, as well as (d) multivalent antigen (2,4,6-trinitrophenyl-bovine serum albumin), all show similar effects and dose-response curves. IgD-negative transgenic B cells are equally sensitive to IgM ligation-dependent inhibition, as control (IgD-positive) B cells. The allotype specificity of this inhibition, assessed by using anti-mu allotype reagents to inhibit and assay the responses, suggests that B cells expressing transgenic or endogenous IgM in transgenic B6-Sp6 mice are largely independent populations. These observations establish that anti-IgM antibodies in conjunction with appropriate LPS stimulation, provide a universal model system for functional characterization of B cell responses.

Research paper thumbnail of Gene expression profiling of erythroblasts from refractory anaemia with ring sideroblasts (RARS) and effects of G-CSF

British Journal of Haematology, 2009

Research paper thumbnail of 5-Fluorouracil-induced RNA stress engages a TRAIL-DISC-dependent apoptosis axis facilitated by p53

Oncotarget, Jan 22, 2015

Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains... more Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-tr...

Research paper thumbnail of cIAP-2 block apoptotic events in bladder cancer cells

Anticancer research

The Inhibitors of Apoptosis (IAPs) are negative regulators of apoptosis and their overexpression ... more The Inhibitors of Apoptosis (IAPs) are negative regulators of apoptosis and their overexpression renders cells resistant to a variety of apoptotic stimuli. We investigated the mRNA expression levels of IAPs in a panel of bladder tumour cells, selected as being sensitive or resistant to death receptor-mediated apoptosis. The mRNA expression of IAPs was quantified in a RNase protection assay (RPA). Apoptosis was induced by recombinant killerTRAIL, agonistic anti-CD95 mAbs or doxorubicin and quantified by TUNEL and MTT assays. Stable expression of cIAP-2 was obtained by retroviral transduction. The expression of cIAP-2 mRNA was highly correlated with resistance to TRAIL-mediated apoptosis. Overexpression of cIAP-2 conferred resistance to previously sensitive cell lines and made cells less susceptible to doxorubicin. Treatment with doxorubicin in combination with TRAIL or anti-CD95 resulted in a synergistic cytotoxic effect, which was not possible to reverse by overexpression of cIAP-2....

Research paper thumbnail of High level of cFLIP correlates with resistance to death receptor-induced apoptosis in bladder carcinoma cells

Anticancer research

The cellular form of FLICE-inhibitory protein (cFLIP) blocks death receptor-induced apoptosis and... more The cellular form of FLICE-inhibitory protein (cFLIP) blocks death receptor-induced apoptosis and has been implicated in tumour progression. cFLIP interacts with caspase-8, thereby preventing activation of the caspase cascade. In this study we investigated the endogenous expression of cFLIP and caspase-8 in bladder carcinoma cells in relation to their sensitivity to death receptor-ligation. Apoptosis was induced by agonistic anti-CD95 mAbs or recombinant TRAIL and quantified by the TUNEL technique. The relative mRNA expression of cFLIP and caspase-8 was quantified by real-time PCR. Stable expression of cFLIP long (cFLIPL) was obtained by retroviral transduction. The relative ratio of cFLIP and caspase-8 was directly correlated to resistance to anti-CD95 or TRAIL-mediated apoptosis. Overexpression of cFLIPL shifted the responsiveness towards resistant status. cFLIP is an important determinant of susceptibility to death receptor-induced apoptosis in bladder carcinomas and could functi...

Research paper thumbnail of Multiparametric analysis of antibody repertoires

Research paper thumbnail of Regulated ChAT journal.pone.0065936

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells... more Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer's disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer's disease and multiple sclerosis in particular.

Research paper thumbnail of Development of B Cells secreting Endogenous or Transgene-Encoded Immunoglobulins in H-Chain Transgenic Mice

Scandinavian Journal of Immunology, 1993

The development of splenic B cells secreting transgene-encoded or endogenous immunoglobulin (Ig) ... more The development of splenic B cells secreting transgene-encoded or endogenous immunoglobulin (Ig) was analysed in the mu heavy (H-)chain transgenic mouse line M54. The results show that cells secreting endogenous Ig are not detectable during the perinatal period, even after lipopolysaccharide stimulation in vitro. At this time, transgene-secreting cells are readily detectable and keep increasing with age of the animals. After a few weeks of age cells secreting endogenous Ig appear in the spleen and keep increasing with age, reaching numbers comparable to non-transgenic littermates by 5 weeks of age. Thereafter, the proportion of transgene-secreting B cells decreases. We conclude that the preferential expression of endogenous Igs by secreting B cells in the adult does not result from peculiar genetic features of those cells, but from age-dependent cellular selection operating on all B cells.

Research paper thumbnail of Angiostatic Effects of NK Cell-Derived IFN- γ Counteracted by Tumour Cell Bcl-x L Expression

Scandinavian Journal of Immunology, 2014

Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the... more Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the antiapoptotic protein Bcl-x L , known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour-progressiveactivity in amurinelymphoma model. Bcl-x L overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl-x L protection of tumours from NK cellmediated attack did not involve protection from NK cell-mediated cytotoxicity. Instead, Bcl-x L -blocked apoptosis resulting from hypoxia and/or nutrient loss associated with theinhibition of angiogenesiscaused by NK cell-secreted IFN-c. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.

Research paper thumbnail of Simultaneous Expression and Detection of Multiple Retroviral Constructs in Haematopoietic Cells After Bone Marrow Transplantation

Scandinavian Journal of Immunology, 2005

Due to the complexity and redundancy of molecular processes governing the development and functio... more Due to the complexity and redundancy of molecular processes governing the development and function of haematopoietic cells, experimental procedures allowing simultaneous alteration in gene expression of multiple genes in vivo are needed. Here, we describe a protocol allowing for simultaneous transduction of haematopoietic stem cells (HSC) with two different replication incompetent retroviral expression vectors followed by transplantation of lethally irradiated recipient mice. These bicistronic retroviral vectors carried genes for the enhanced green and yellow florescent proteins (EGFP and EYFP) respectively. Spleen cells from reconstituted animals were stained for common lymphocyte and myeloid markers, then analysed on a two-laser, 488 and 635 nm, flow cytometer equipped with a 510/20-nm bandpass filter for FL1, a 550/30-nm bandpass filter for FL2 and a 530-nm short-pass dichroic mirror. It was demonstrated that cells expressing EGFP, EYFP or combinations thereof could be distinguished and analysed for staining with PerCP-and APC-conjugated reagents. We found that a sizable proportion of cells (70%) from reconstituted animals expressed EGFP and/or EYFP and that expression of these genes did not affect lymphoid or myeloid development. We also demonstrated that the alternative optical configuration allowed for conventional multiparameter flow cytometric analyses.

Research paper thumbnail of Participation of FLIP, RIP and Bcl-x L in Fas-mediated T-cell Death

Scandinavian Journal of Immunology, 2007

Apart from the conventional Fas signalling pathway, alternative pathways including the mitochondr... more Apart from the conventional Fas signalling pathway, alternative pathways including the mitochondrial caspase-dependent and RIP-mediated cell death routes have been proposed to operate during Fas-mediated cell death. To evaluate the contribution of different Fas signalling pathways, mice overexpressing FLIP(L), Bcl-x(L), a kinase-deficient form of RIP (RIPDeltakin) or combinations thereof were generated by retroviral gene transfer of haematopoietic stem cells. Such mice did not show overt abnormalities in haematopoietic development, defects in thymic deletion, accumulation of double-negative T cells or signs of autoimmunity. Fas-mediated death of mitogen-activated T cells was caspase dependent and could be blocked by FLIP(L) overexpression only with the minor involvement of Bcl-x(L) or RIPDeltakin inhibitable pathways. Fas-mediated death of resting CD4(+) and CD8(+) T cells was mainly caspase dependent but could only partly be blocked by FLIP(L) overexpression. Both Bcl-x(L) or RIPDeltakin expression resulted in partial protection of CD8(+) T cells against Fas-mediated cell death. These results indicate that yet uncharacterized signalling pathways from the Fas receptor are critically involved in lymphoproliferative and autoimmune disease observed in lpr mice and autoimmune lymphoproliferative syndrome patients.

Research paper thumbnail of Acquisition of CD40 Expression During Murine B-Cell Differentiation

Scandinavian Journal of Immunology, 1996

Expression of CD40 on mouse cells was investigated comparing the binding to cells of a monoclonal... more Expression of CD40 on mouse cells was investigated comparing the binding to cells of a monoclonal antibody against CD40, to that of a soluble fusion protein consisting of the extracellular domains of the mouse CD40-ligand. The analysis of a series of established cell lines failed to demonstrate expression of CD40 on pro- or pre-B cells, and indicated that CD40 expression was restricted to cells that had undergone productive heavy- and light-chain gene rearrangements, and expressed surface Ig. In cells from normal mice, CD40 first becomes detectable, although at low levels, on a subset of small pre-B-II cells in bone marrow, the levels of CD40 expression increasing thereafter during B-cell maturation. Thus, immature B cells (IgM+ IgDlo B220lo) express intermediate levels of CD40, and mature B cells (IgM+ IgDhi B220hi) express high levels of CD40. Anatomical location also seems to correlate with the levels of CD40 expression, as B cells expressing the highest levels of CD40 were found in lymph nodes and Peyer's patches.

Research paper thumbnail of Inhibition of the Intrinsic but Not the Extrinsic Apoptosis Pathway Accelerates and Drives Myc-Driven Tumorigenesis Towards Acute Myeloid Leukemia

PLoS ONE, 2012

Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However... more Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However, MYC is also a powerful inducer of apoptosis, which is one of the major failsafe programs to prevent cancer development. To clarify the relative importance of the extrinsic (death receptor-mediated) versus the intrinsic (mitochondrial) pathway of apoptosis in MYC-driven AML, we coexpressed MYC together with anti-apoptotic proteins of relevance for AML; BCL-X(L)/BCL-2 (inhibiting the intrinsic pathway) or FLIP(L) (inhibiting the extrinsic pathway), in hematopoietic stems cells (HSCs). Transplantation of HSCs expressing MYC into syngeneic recipient mice resulted in development of AML and T-cell lymphomas within 7-9 weeks as expected. Importantly, coexpression of MYC together with BCL-X(L)/BCL-2 resulted in strongly accelerated kinetics and favored tumor development towards aggressive AML. In contrast, coexpression of MYC and FLIP(L) did neither accelerate tumorigenesis nor change the ratio of AML versus T-cell lymphoma. However, a change in distribution of immature CD4(+)CD8(+) versus mature CD4(+) T-cell lymphoma was observed in MYC/FLIP(L) mice, possibly as a result of increased survival of the CD4+ population, but this did not significantly affect the outcome of the disease. In conclusion, our findings provide direct evidence that BCL-X(L) and BCL-2 but not FLIP(L) acts in synergy with MYC to drive AML development.

Research paper thumbnail of DISC-mediated activation of caspase-2 in DNA damage-induced apoptosis

Oncogene, 2009

The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signal... more The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signaling cascade regulated by sequential activation of caspases. Mechanisms that lead from p53 to activation of individual initiator caspases are still unclear. The present model for caspase-2 activation includes PIDDosome complex formation. However, in certain experimental models, elimination of complex constituents PIDD or RAIDD did not significantly influence caspase-2 activation, suggesting the existence of an alternative activation platform for caspase-2. Here we have investigated the link between p53 and caspase-2 in further detail and report that the latter is able to utilize the CD95 DISC as an activation platform. The recruitment of caspase-8 to this complex is required for activation of caspase-2. In the experimental system used, the DISC is formed through a distinct, p53-dependent upregulation of CD95. Moreover, we show that caspase-2 and -8 cleave Bid, and that both act simultaneously upstream of mitochondrial cytochrome c release. Finally, a direct interaction between the two caspases and the ability of caspase-8 to cleave caspase-2 are demonstrated. Thus, the observed functional link between caspase-8 and -2 within the DISC represents an alternative mechanism to the PIDDosome for caspase-2 activation in response to DNA damage.

Research paper thumbnail of Host strain-dependent difference in susceptibility in a rat model of herpes simplex type 1 encephalitis

Journal of Neurovirology, 2008

Herpes simplex encephalitis (HSE) is characterized by severe focal brain inflammation leading to ... more Herpes simplex encephalitis (HSE) is characterized by severe focal brain inflammation leading to substantial loss of nervous tissue. The authors established a model of Herpes simplex virus type 1 (HSV)-1-induced acute encephalitis in the rat by injecting into the whiskers' area a virus strain isolated from a fatal human HSE case. The model might resemble natural propagation of HSV-1 in humans; spreading from the mouth and lips via the trigeminal nerve to trigeminal ganglia and subsequently entering the central nervous system (CNS). HSV-1 infected Dark Agouti (DA) rats developed a well-synchronized disease and died 5 days after inoculation. HSV-1 detection by quantitative polymerase chain reaction (qPCR), virus isolation and immunohistochemistry, magnetic resonance imaging, and histopathological examination verified dramatic encephalitis mainly in the brainstem, but also in the olfactory bulb and other segments of the brain of diseased rats. In contrast, Piebald Virol Glaxo (PVG) rats were completely resistant to disease, displaying a more rapid clearance of peripheral infection and no evidence of virus entering into neither the trigeminal ganglia nor the CNS. These results suggest a regulation of susceptibility to HSV-1-induced encephalitis at the level of peripheral infection and subsequent neuronal uptake/transport of the virus. This provides a basis for future positioning of genetic polymorphisms regulating HSE and for dissection of important pathogenetic mechanisms of this severe human disease.

Research paper thumbnail of Expression of the Long Form of Human FLIP by Retroviral Gene Transfer of Hemopoietic Stem Cells Exacerbates Experimental Autoimmune Encephalomyelitis

The Journal of Immunology, 2003

To test this concept, we examined the effects of overexpressing the long form of human FLICEinhib... more To test this concept, we examined the effects of overexpressing the long form of human FLICEinhibitory protein, a potent inhibitor of death receptor-mediated apoptosis, in myelin oligodendrocyte glycoprotein-induced EAE in DBA/1 mice. We found that overexpression of the long form of human FLICE-inhibitory protein by retroviral gene transfer of hemopoietic stem cells led to a clinically more severe EAE in these mice compared with control mice receiving the retroviral vector alone. The exacerbated disease was evident by an enhanced and prolonged inflammatory reaction in the CNS of these animals compared with control mice.

Research paper thumbnail of Death Receptor Ligation or Exposure to Perforin Trigger Rapid Egress of the Intracellular Parasite Toxoplasma gondii

The Journal of Immunology, 2007

Research paper thumbnail of Expression and Selection of Murine Antibody Repertoires

International Reviews of Immunology, 1992

Research paper thumbnail of Endogenous V H gene family expression in immunoglobulin-transgenic mice: evidence for selection of antibody repertoires

International Immunology, 1991

VH gene family expression in single cells of the emergent, available and actual B cell repertoire... more VH gene family expression in single cells of the emergent, available and actual B cell repertoires of C57BL/6 mice was compared to that of two immunoglobulin (Ig)-transgenic B6 lines (B6-Sp6 and M54). We found that less than 5% of bone marrow cells of transgenic mice express endogenous VH genes and that the vast majority (95%) of the peripheral, mature B cell repertoire in these animals is composed of cells expressing the VHJ558 transgenic family. Unimmunized transgenic mice, however, diversify VH gene family usage by 'background' Ig-secreting cells in the spleen, greater than 50% of which express endogenous VH genes. The pattern of endogenous VH gene family expression in the actual repertoire of B6-Sp6 mice is indistinguishable from that of normal B6 mice. In contrast, actual repertoires of M54 mice differ by a 4- to 5-fold higher representation of the VHQ52 family. These results demonstrate a powerful positive selection of B cells into the secretory compartments of unimmunized animals, show that actual and available repertoires differ very markedly, and suggest that V region interactions participate in the selection of 'natural antibody' repertoires.

Research paper thumbnail of An example of idiotypic mimicry

European Journal of Immunology, 1997

We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibod... more We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibody repertoires. The transgenic system was chosen as to allow for normal recombination of endogenous Ig genes, secretion of TGIg from early development on, and distinguishing the TGIg from endogenous Ig by several serological markers on the C and V regions of the molecules. The transgenic construct encodes a complete anti-(4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NP) antibody molecule carrying a well-defined idiotype, bearing a lambda 1 light chain and a chimeric heavy chain encoded by a human alpha 2 C region devoid of its membrane exon, and the murine B1.8 VDJ-region. Endogenous antibody repertoires were analyzed in mitogen-driven limiting dilution cultures, in single-cell assays for naturally activated Ig-secreting cells, and in hybridomas derived by direct fusion of spleen cells from unmanipulated animals. The results show that a very high frequency of splenic resting B cells and plasma cells in transgenic animals produce IgM with B1.8-cross-reactive idiotypes. This was confirmed by hybridoma analysis which also established that the levels of transgene expression and of idiotype-positive IgM production by the same cell are not correlated. The affinities of idiotype-positive endogenous Ig varied, but were generally several orders of magnitude lower than the transgene-encoded idiotype. V regions from idiotype-cross-reactive IgM heavy chains showed marked diversity in sequences that were all different from the transgenic B1.8. These results are compatible with idiotypic mimicry resulting from intercellular selection based on degenerate, whole V region reactivities.

Research paper thumbnail of Suppression of B cell differentiation by ligation of membrane-bound IgM

European Journal of Immunology, 1993

Using B cells from the transgenic mouse line B6-Sp6 and control littermates, stimulated by lipopo... more Using B cells from the transgenic mouse line B6-Sp6 and control littermates, stimulated by lipopolysaccharide (LPS) under novel culture conditions that provide for the response of all B cells, we show here that specific ligation of the surface IgM molecules always results in inhibition of terminal differentiation and immunoglobulin secretion by activated cells, regardless of the ligand. Thus, monoclonal antibodies to (a) the CH region of Ig (anti-mu and anti-allotype), (b) the C kappa region, (c) the V region (anti-idiotype) of surface IgM, as well as (d) multivalent antigen (2,4,6-trinitrophenyl-bovine serum albumin), all show similar effects and dose-response curves. IgD-negative transgenic B cells are equally sensitive to IgM ligation-dependent inhibition, as control (IgD-positive) B cells. The allotype specificity of this inhibition, assessed by using anti-mu allotype reagents to inhibit and assay the responses, suggests that B cells expressing transgenic or endogenous IgM in transgenic B6-Sp6 mice are largely independent populations. These observations establish that anti-IgM antibodies in conjunction with appropriate LPS stimulation, provide a universal model system for functional characterization of B cell responses.