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Expression of HMGA2 in blood cells from patients with paroxysmal nocturnal haemoglobinuria
Abstracts / Molecular Immunology 45 (2008) 4095–4182
trols and C3 showed a weak correlation with fH (r2 = 0.253). Serum fH may be a useful biomarker in MS, particularly for determining relapse status and disease course and providing objective evidence to help guide therapeutic decisions. The data also implicates activation of the alternative pathway in chronic MS. doi:10.1016/j.molimm.2008.08.244 P246 A novel concept for the treatment of paroxysmal nocturnal haemoglobinuria (PNH): Complement depletion with a human C3 derivative with cobra venom factor-like activity prevents lysis of PNH erythrocytes David Fritzinger a , Viviana Ferreira b , Brian Hew a , William St. John c , Michael Pangburn b , Carl-Wilhelm Vogel a a
Cancer Research Center of Hawaii, Honolulu, USA University of Texas Health Science Center, Tyler, USA c InCode Biopharmaceuticals Inc., San Francisco, USA b
PNH is a rare chronic blood disorder characterized by complement-mediated lysis of red blood cells that partially (Type II) or completely (Type III) lack the regulatory proteins CD59 and CD55. The only treatment currently available for PNH patients is maintenance therapy with an anti-C5 antibody (eculizumabTM). Here we report complement depletion with a human C3 derivative with cobra venom factor (CVF)-like activity (HC3-1496) as a novel therapeutic concept for PNH. HC3-1496 is a recombinant human C3/CVF hybrid protein in which the 168 C-terminal amino acids of C3 have been replaced with the corresponding amino acids from CVF. Normal human serum (NHS) was treated with HC3-1496 to either achieve complete complement depletion or a reduction of serum complement activity by only 33%. Subsequently, erythrocytes from 4 PNH patients were incubated with the treated serum samples in the absence or presence of a recombinant protein encompassing the C-terminus of factor H (rH19-20) which blocks the cell-surface complement regulatory functions of factor H, making PNH Type II and III cells highly susceptible to complement-mediated lysis in NHS. After incubation, the CD59 profile of the remaining unlysed cells was determined by FACS analysis with an FITC-conjugated anti-CD59 monoclonal antibody from which the percent lysis of PNH cells was calculated [Blood 110, 2007, 2190–2192]. HC3-1496 treatments yielded serum samples exhibiting no or as much as 67% remaining complement activity and prevented lysis of the PNH cells from all four patients. NHS and NHS complement-depleted with CVF served as positive and negative controls, respectively. In conclusion, complement depletion with HC3-1496 appears to be a promising treatment of PNH. It is important to note that HC3-1496 does not consume C5, leaving its protective function against serious meningococcal infections in place. doi:10.1016/j.molimm.2008.08.245
4177
P247 Expression of HMGA2 in blood cells from patients with paroxysmal nocturnal haemoglobinuria Yoshiko Murakami a , Rieko Ohta b , Norimitsu Inoue c , Junichi Nishimura a , Yuzuru Kanakura a , Taroh Kinoshita a a
Osaka University, Suita, Japan Nagoya University, Nagoya, Japan c Osaka Medical Center, Osaka, Japan b
Paroxysmal nocturnal hemoglobinuria (PNH) is a consequence of clonal expansion of hematopoietic stem cells with somatic mutation of PIG-A. We are proposing a 3-step model of PNH pathogenesis. Step 1 involves the somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative advantage. According to this hypothesis, we searched for the candidate gene for Step 3. We reported two patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome 12, making the break within the 3 f untranslated region of HMGA2. This is the architectural transcription factor gene deregulated in many benign mesenchymal tumours [Blood 108(13) 2006, 4232]. Recently, many reports show that truncation of 3 f untranslated region of HMGA2 disrupts binding of miRNA, let-7 which regulates both transcription and translation. Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation. We have established the method for stable isolation of mRNA and miRNA from blood and bone marrow cells from PNH patients and analyzed the expression of HMGA2 and let-7 by quantitative RT-PCR. We have analyzed the peripheral blood from 10 healthy volunteers and 3 PNH patients including one patient with chromosomal abnormality as a positive control. The sample from one patient without chromosomal abnormality had significantly higher expression of HMGA2. We analyzed the genomic sequence of this patient and found no mutation in 3 f untranslated region. We also analyzed the expression of miRNA and found decreased expression of let7a-e in this patient. We are going to analyze more PNH samples to investigate whether high expression of HMGA2 contributes the pathogenesis of PNH. doi:10.1016/j.molimm.2008.08.246 P248 Impact of oral contraceptives containing different progestagens and ethinylestradiol on the hemolytic activity of the complement system Suellen Priscila dos Santos Migueleti, Ana Elisa Caleiro Seixas Azzolini, Yara Maria Lucisano-Valim, Maria Regina Torqueti Toloi, Luciana Simon Pereira Crott, Cleni Mara Marzocchi-Machado Fac. Ciênc Farmacêuticas-Rib Preto-USP, Ribeirão Preto, Brazil Estrogen, as oral contraceptives (OC), is associated with the thromboembolic and chronic inflammatory diseases. It is observed an increase of the factor XII, decrease of the S protein and a clinical manifestation similar to the hereditary angioedema. C1 inhibitor regulates the factor XII and its decrease can result in the increase of factor XII, generation of the bradykinin and C2 proteolysis by the classical pathway (CP), leading to angioedema. Since the complement and the coagulation are related physiological systems, there is an interest in the estrogen effects on these systems. This study investigated the influence of the OC with different progestagens and ethinylestradiol concentrations on the hemolytic activity
trols and C3 showed a weak correlation with fH (r2 = 0.253). Serum fH may be a useful biomarker in MS, particularly for determining relapse status and disease course and providing objective evidence to help guide therapeutic decisions. The data also implicates activation of the alternative pathway in chronic MS. doi:10.1016/j.molimm.2008.08.244 P246 A novel concept for the treatment of paroxysmal nocturnal haemoglobinuria (PNH): Complement depletion with a human C3 derivative with cobra venom factor-like activity prevents lysis of PNH erythrocytes David Fritzinger a , Viviana Ferreira b , Brian Hew a , William St. John c , Michael Pangburn b , Carl-Wilhelm Vogel a a
Cancer Research Center of Hawaii, Honolulu, USA University of Texas Health Science Center, Tyler, USA c InCode Biopharmaceuticals Inc., San Francisco, USA b
PNH is a rare chronic blood disorder characterized by complement-mediated lysis of red blood cells that partially (Type II) or completely (Type III) lack the regulatory proteins CD59 and CD55. The only treatment currently available for PNH patients is maintenance therapy with an anti-C5 antibody (eculizumabTM). Here we report complement depletion with a human C3 derivative with cobra venom factor (CVF)-like activity (HC3-1496) as a novel therapeutic concept for PNH. HC3-1496 is a recombinant human C3/CVF hybrid protein in which the 168 C-terminal amino acids of C3 have been replaced with the corresponding amino acids from CVF. Normal human serum (NHS) was treated with HC3-1496 to either achieve complete complement depletion or a reduction of serum complement activity by only 33%. Subsequently, erythrocytes from 4 PNH patients were incubated with the treated serum samples in the absence or presence of a recombinant protein encompassing the C-terminus of factor H (rH19-20) which blocks the cell-surface complement regulatory functions of factor H, making PNH Type II and III cells highly susceptible to complement-mediated lysis in NHS. After incubation, the CD59 profile of the remaining unlysed cells was determined by FACS analysis with an FITC-conjugated anti-CD59 monoclonal antibody from which the percent lysis of PNH cells was calculated [Blood 110, 2007, 2190–2192]. HC3-1496 treatments yielded serum samples exhibiting no or as much as 67% remaining complement activity and prevented lysis of the PNH cells from all four patients. NHS and NHS complement-depleted with CVF served as positive and negative controls, respectively. In conclusion, complement depletion with HC3-1496 appears to be a promising treatment of PNH. It is important to note that HC3-1496 does not consume C5, leaving its protective function against serious meningococcal infections in place. doi:10.1016/j.molimm.2008.08.245
4177
P247 Expression of HMGA2 in blood cells from patients with paroxysmal nocturnal haemoglobinuria Yoshiko Murakami a , Rieko Ohta b , Norimitsu Inoue c , Junichi Nishimura a , Yuzuru Kanakura a , Taroh Kinoshita a a
Osaka University, Suita, Japan Nagoya University, Nagoya, Japan c Osaka Medical Center, Osaka, Japan b
Paroxysmal nocturnal hemoglobinuria (PNH) is a consequence of clonal expansion of hematopoietic stem cells with somatic mutation of PIG-A. We are proposing a 3-step model of PNH pathogenesis. Step 1 involves the somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative advantage. According to this hypothesis, we searched for the candidate gene for Step 3. We reported two patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome 12, making the break within the 3 f untranslated region of HMGA2. This is the architectural transcription factor gene deregulated in many benign mesenchymal tumours [Blood 108(13) 2006, 4232]. Recently, many reports show that truncation of 3 f untranslated region of HMGA2 disrupts binding of miRNA, let-7 which regulates both transcription and translation. Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation. We have established the method for stable isolation of mRNA and miRNA from blood and bone marrow cells from PNH patients and analyzed the expression of HMGA2 and let-7 by quantitative RT-PCR. We have analyzed the peripheral blood from 10 healthy volunteers and 3 PNH patients including one patient with chromosomal abnormality as a positive control. The sample from one patient without chromosomal abnormality had significantly higher expression of HMGA2. We analyzed the genomic sequence of this patient and found no mutation in 3 f untranslated region. We also analyzed the expression of miRNA and found decreased expression of let7a-e in this patient. We are going to analyze more PNH samples to investigate whether high expression of HMGA2 contributes the pathogenesis of PNH. doi:10.1016/j.molimm.2008.08.246 P248 Impact of oral contraceptives containing different progestagens and ethinylestradiol on the hemolytic activity of the complement system Suellen Priscila dos Santos Migueleti, Ana Elisa Caleiro Seixas Azzolini, Yara Maria Lucisano-Valim, Maria Regina Torqueti Toloi, Luciana Simon Pereira Crott, Cleni Mara Marzocchi-Machado Fac. Ciênc Farmacêuticas-Rib Preto-USP, Ribeirão Preto, Brazil Estrogen, as oral contraceptives (OC), is associated with the thromboembolic and chronic inflammatory diseases. It is observed an increase of the factor XII, decrease of the S protein and a clinical manifestation similar to the hereditary angioedema. C1 inhibitor regulates the factor XII and its decrease can result in the increase of factor XII, generation of the bradykinin and C2 proteolysis by the classical pathway (CP), leading to angioedema. Since the complement and the coagulation are related physiological systems, there is an interest in the estrogen effects on these systems. This study investigated the influence of the OC with different progestagens and ethinylestradiol concentrations on the hemolytic activity