<p>Immunohistochemistry was used to localize PDGF-CC expression using 6B3 antibody in selec... more <p>Immunohistochemistry was used to localize PDGF-CC expression using 6B3 antibody in selected human control tissues. A) In the positive control (A459 cell line) staining with mAb 6B3 showed strong PDGF-CC expression (left panel) whereas no PDGF-CC expression could be detected upon omission of the primary antibody (2<sup>nd</sup> panel to the right) or when the primary antibody was incubated with excess PDGF-CC protein (2<sup>nd</sup> panel to the left), in contrast to incubation with an unspecific antigen (right panel). B) Representative images using 6B3 or CD34 antibodies in 10 control human tissues showed the differential expression level of PDGF-CC in various organs (see comments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201089#pone.0201089.t003" target="_blank">Table 3</a>). CD34 is used to show the outline of the blood vessels. Magnification 200X.</p
<p>A) 6B3 recognizes the reduced (R) and non-reduced (NR) human PDGF-CC protein both the ac... more <p>A) 6B3 recognizes the reduced (R) and non-reduced (NR) human PDGF-CC protein both the active (hPCc) and latent (hPCf) PDGF-CC in WB assay, but does neither recognize active (mPCc) mouse PDGF-CC, nor active human PDGF-DD (hPDc). B) Biosensor analysis of the apparent binding affinity. The apparent on-rate (ka), off-rate (kd) and dissociation constants are shown. The calculated dissociation constants for each antibody were in the low nano molar range, indicating that all 3 antibodies had a high affinity for the PDGF-CC ligand. C) Cross-competition for binding of mAbs to immobilized PDGF-CC. The effectiveness of the mAbs to cross compete for binding to immobilized PDGF-CC ligand is shown: Green: binding antibodies (different epitope); Red: competing antibodies (similar epitope); Blue: antibodies that interfere with binding. D) PAE1-PDGFRα cells were stimulated with either 30 ng hPCc, 6B3, or a mixture of 30 ng hPCc and preincubated 6B3 together for 1 h. PY99 was used to examine the phosphorylation status of PDGFRα. Only cells stimulated with PDGF-CC alone showed strong phosphorylation of PDGFRα, and this activation was blocked when 6B3 was pre-incubated together with the ligand (upper panel). The receptor expression level was detected with anti-CED (antibody against PDGFRs) and served as control. E) 96-well plate was coated with PDGF-AA, PDGF-BB, PDGF-CC or PDGF-DD and ELISA showed that 6B3 recognized only PDGF-CC but not other PDGF ligands. The value is the 405nM absorbance from the average of 8 samples ± standard deviation. F) Quality control for the PDGF ligands: 96-well plate was coated with recombinant PDGFRα protein or PDGF-DD and ELISA showed that PDGF-AA, PDGF-BB and PDGF-CC, but not PDGF-DD could detect PDGFRα. K618, a rabbit polyclonal antibody against PDGF-DD was used to assess protein quality of PDGF-DD. NC (not coated).</p
<p>A) Multiple Fragment Molecular Dynamics (MFMD) was used for modelling and prediction of ... more <p>A) Multiple Fragment Molecular Dynamics (MFMD) was used for modelling and prediction of the epitope for 6B3. The 3D structure of the activated PDGF-CC dimer showed 4 binding sites that are indicated with different colours. B) Amino acid sequence of activated PDGF-CC and the predicted peptides. C) Biocore analysis showed that peptide number 3 is competing with activated PDGF-CC for binding to 6B3 when 6B3 was pre-incubated with peptide 3, but not with any other peptides. D) Dose dependent binding of the immobilized peptide 3 to 6B3.</p
&amp;lt;p&amp;gt;A) CDRs of the ch6B3 are indicated. B) Chimeric 6B3 (ch6B3) specifically... more &amp;lt;p&amp;gt;A) CDRs of the ch6B3 are indicated. B) Chimeric 6B3 (ch6B3) specifically recognizes hPDGF-CC under both reduced (R) and non-reduced (NR) conditions, but does not recognize mouse (mPDGF-CC) and rat (rPDGF-CC) PDGF-CC in immunoblots. C) ELISA showed that ch6B3 specifically detected PDGF-CC, but not any other PDGF ligands. D) Using PAE1-PDGFRα cells ch6B3 showed equal neutralizing capacity in comparison with mouse 6B3 in PDGF-CC induced phosphorylation of PDGFRα. E) Left panel: Co-staining for PDGFRα (in red) and the endothelial marker podocalyxin (in green) in whole-mount retina. Arrow heads point to perivascular PDGFRα+ cells ensheathing podocalyxin+ vessels (left panel). 50μm scale bar. Right panel: Co-staining for astrocyte marker GFAP (in red) in retina cross-section from &amp;lt;i&amp;gt;Pdgfrα&amp;lt;/i&amp;gt;&amp;lt;sup&amp;gt;&amp;lt;i&amp;gt;GFP/+&amp;lt;/i&amp;gt;&amp;lt;/sup&amp;gt; (in green) reporter mouse. Arrow heads point to double-positive cells in the ganglion cell layer, confirming that perivascular astrocytes in retina express PDGFRα 10 μm scale bar. F) Intraocular injection of PDGF-CC caused extravasation of the plasma-derived tracer TMR-Dex (red) in an &amp;lt;i&amp;gt;in vivo&amp;lt;/i&amp;gt; BRB assay. BRB disruption was abrogated by intraperitoneal injection of ch6B3. Blood vessels were visualized by staining against Podocalyxin. Representative images are shown, based on n = 4 mice injected with isotype control Ig, and n = 4 mice injected with ch6b3 antibody. Scale bar 10 μm. G) Quantification of vascular permeability based on red fluorescent pixel area recorded in retina whole-mounts (n = 4 mice/experimental group). Abbreviations: hPCc: human PDGF-CC, GCL: ganglion cell layer, ICL: inner cell layer.&amp;lt;/p
<p>Organs with significant higher PDGF-CC expression in tumour than their respective contro... more <p>Organs with significant higher PDGF-CC expression in tumour than their respective control tissues.</p
<p>Representative images using 6B3 and CD34 antibody in bladder transitional cell carcinoma... more <p>Representative images using 6B3 and CD34 antibody in bladder transitional cell carcinoma (TCC), brain glioblastoma, breast infiltrating duct carcinoma (IDC), kidney renal cell carcinoma (RCC), pancreas and prostate adenocarcinoma (Adca) showed significantly higher expression level of PDGF-CC in comparison to their control tissues (see comments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201089#pone.0201089.t004" target="_blank">Table 4</a>), magnification 200X.</p
PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα ph... more PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα phosphorylation and thereby activates intracellular signalling events essential for development but also in cancer, fibrosis and neuropathologies involving blood-brain barrier (BBB) disruption. In order to elucidate the biological and pathological role(s) of PDGF-CC signalling, we have generated high affinity neutralizing monoclonal antibodies (mAbs) recognizing human PDGF-CC. We determined the complementarity determining regions (CDRs) of the selected clones, and mapped the binding epitope for clone 6B3. Using the monoclonal 6B3, we determined the expression pattern for PDGF-CC in different human primary tumours and control tissues, and explored its ability to neutralize PDGF-CC-induced phosphorylation of PDGFRα. In addition, we showed that PDGF-CC induced disruption of the blood-retinal barrier (BRB) was significantly reduced upon intraperitoneal administration of a chimeric anti-PDGF-CC antibody. In summary, we report on high affinity monoclonal antibodies against PDGF-CC that have therapeutic efficacy in vivo.
It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly... more It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2...
It is demonstrated, using two different perfusion reaction systems, that hybridoma modified by in... more It is demonstrated, using two different perfusion reaction systems, that hybridoma modified by inhibiting their apoptotic response can give improved process performance in terms of cell number and viability in intensive cell culture. Two cell perfusion systems, one using a spin filter and the other an ultrasonic filter, are compared using two cell lines. One cell line is transfected with the bcl-2 gene (TB/C3 bcl-2) which encodes the 'anti-apoptotic' human bcl-2 protein and the other cell line (TB/C3 pEF) with a negative transfection vector. Both reactor systems give similar retention performance for both cell lines. Bcl-2 transfected cells reach higher cell densities than the control cell line, and the percentage of apoptotic cells is clearly lower than with pEF cells. The maximum cell numbers of the bcl-2 cell line are 1.21× 10 7 ml − 1 in the ultrasonic filter culture and 1.58× 10 7 ml − 1 in the spin filter culture, respectively. Using the pEF cell line the maximum cell number reaches 6.0 × 10 6 ml − 1 with ultrasonic retention and 5.9 × 10 6 ml − 1 in the spin filter. The use of ultrasound in this cell retention system has no apparent influence on cell growth, productivity or viability. Selective retention of viable cells is detectable but the effect of removing non-viable cells is negligible.
This report represents an investigation into the nature of apoptosis in hybridoma cultures and it... more This report represents an investigation into the nature of apoptosis in hybridoma cultures and its significance to their utilization in biotechnology. To this end DNA fragmentation and capillary electrophoresis of genomic DNA was studied during the culture of two hybridoma cell lines. This indicated that the phenomenon of apoptosis was always present even under normal culture conditions. Two DNA fragments not associated with the typical DNA fragmentation ladder were identified in the two hybridoma cultures: a previously unreported DNA fragment of about 100 bp and a large fragment which may correspond to one reported in the literature (Walker et al., 1993). The small fragment was identified as soon as the early exponential growth phase of culture, while the large fragment appeared only in the latter part of the growth curve when there was marked DNA fragmentation. In addition we present evidence that aurintricarboxyiic acid, which inhibits apoptosis in neural cells, permits this process in hybridoma cells at levels below 100 PM. This unusual predisposition of hybridoma cultures to undergo apoptosis and their response to inhibitor of apoptosis may have important implications for approaches to the culture of hybridomas and their utilization for monoclonal production.
<p>Immunohistochemistry was used to localize PDGF-CC expression using 6B3 antibody in selec... more <p>Immunohistochemistry was used to localize PDGF-CC expression using 6B3 antibody in selected human control tissues. A) In the positive control (A459 cell line) staining with mAb 6B3 showed strong PDGF-CC expression (left panel) whereas no PDGF-CC expression could be detected upon omission of the primary antibody (2<sup>nd</sup> panel to the right) or when the primary antibody was incubated with excess PDGF-CC protein (2<sup>nd</sup> panel to the left), in contrast to incubation with an unspecific antigen (right panel). B) Representative images using 6B3 or CD34 antibodies in 10 control human tissues showed the differential expression level of PDGF-CC in various organs (see comments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201089#pone.0201089.t003" target="_blank">Table 3</a>). CD34 is used to show the outline of the blood vessels. Magnification 200X.</p
<p>A) 6B3 recognizes the reduced (R) and non-reduced (NR) human PDGF-CC protein both the ac... more <p>A) 6B3 recognizes the reduced (R) and non-reduced (NR) human PDGF-CC protein both the active (hPCc) and latent (hPCf) PDGF-CC in WB assay, but does neither recognize active (mPCc) mouse PDGF-CC, nor active human PDGF-DD (hPDc). B) Biosensor analysis of the apparent binding affinity. The apparent on-rate (ka), off-rate (kd) and dissociation constants are shown. The calculated dissociation constants for each antibody were in the low nano molar range, indicating that all 3 antibodies had a high affinity for the PDGF-CC ligand. C) Cross-competition for binding of mAbs to immobilized PDGF-CC. The effectiveness of the mAbs to cross compete for binding to immobilized PDGF-CC ligand is shown: Green: binding antibodies (different epitope); Red: competing antibodies (similar epitope); Blue: antibodies that interfere with binding. D) PAE1-PDGFRα cells were stimulated with either 30 ng hPCc, 6B3, or a mixture of 30 ng hPCc and preincubated 6B3 together for 1 h. PY99 was used to examine the phosphorylation status of PDGFRα. Only cells stimulated with PDGF-CC alone showed strong phosphorylation of PDGFRα, and this activation was blocked when 6B3 was pre-incubated together with the ligand (upper panel). The receptor expression level was detected with anti-CED (antibody against PDGFRs) and served as control. E) 96-well plate was coated with PDGF-AA, PDGF-BB, PDGF-CC or PDGF-DD and ELISA showed that 6B3 recognized only PDGF-CC but not other PDGF ligands. The value is the 405nM absorbance from the average of 8 samples ± standard deviation. F) Quality control for the PDGF ligands: 96-well plate was coated with recombinant PDGFRα protein or PDGF-DD and ELISA showed that PDGF-AA, PDGF-BB and PDGF-CC, but not PDGF-DD could detect PDGFRα. K618, a rabbit polyclonal antibody against PDGF-DD was used to assess protein quality of PDGF-DD. NC (not coated).</p
<p>A) Multiple Fragment Molecular Dynamics (MFMD) was used for modelling and prediction of ... more <p>A) Multiple Fragment Molecular Dynamics (MFMD) was used for modelling and prediction of the epitope for 6B3. The 3D structure of the activated PDGF-CC dimer showed 4 binding sites that are indicated with different colours. B) Amino acid sequence of activated PDGF-CC and the predicted peptides. C) Biocore analysis showed that peptide number 3 is competing with activated PDGF-CC for binding to 6B3 when 6B3 was pre-incubated with peptide 3, but not with any other peptides. D) Dose dependent binding of the immobilized peptide 3 to 6B3.</p
&amp;lt;p&amp;gt;A) CDRs of the ch6B3 are indicated. B) Chimeric 6B3 (ch6B3) specifically... more &amp;lt;p&amp;gt;A) CDRs of the ch6B3 are indicated. B) Chimeric 6B3 (ch6B3) specifically recognizes hPDGF-CC under both reduced (R) and non-reduced (NR) conditions, but does not recognize mouse (mPDGF-CC) and rat (rPDGF-CC) PDGF-CC in immunoblots. C) ELISA showed that ch6B3 specifically detected PDGF-CC, but not any other PDGF ligands. D) Using PAE1-PDGFRα cells ch6B3 showed equal neutralizing capacity in comparison with mouse 6B3 in PDGF-CC induced phosphorylation of PDGFRα. E) Left panel: Co-staining for PDGFRα (in red) and the endothelial marker podocalyxin (in green) in whole-mount retina. Arrow heads point to perivascular PDGFRα+ cells ensheathing podocalyxin+ vessels (left panel). 50μm scale bar. Right panel: Co-staining for astrocyte marker GFAP (in red) in retina cross-section from &amp;lt;i&amp;gt;Pdgfrα&amp;lt;/i&amp;gt;&amp;lt;sup&amp;gt;&amp;lt;i&amp;gt;GFP/+&amp;lt;/i&amp;gt;&amp;lt;/sup&amp;gt; (in green) reporter mouse. Arrow heads point to double-positive cells in the ganglion cell layer, confirming that perivascular astrocytes in retina express PDGFRα 10 μm scale bar. F) Intraocular injection of PDGF-CC caused extravasation of the plasma-derived tracer TMR-Dex (red) in an &amp;lt;i&amp;gt;in vivo&amp;lt;/i&amp;gt; BRB assay. BRB disruption was abrogated by intraperitoneal injection of ch6B3. Blood vessels were visualized by staining against Podocalyxin. Representative images are shown, based on n = 4 mice injected with isotype control Ig, and n = 4 mice injected with ch6b3 antibody. Scale bar 10 μm. G) Quantification of vascular permeability based on red fluorescent pixel area recorded in retina whole-mounts (n = 4 mice/experimental group). Abbreviations: hPCc: human PDGF-CC, GCL: ganglion cell layer, ICL: inner cell layer.&amp;lt;/p
<p>Organs with significant higher PDGF-CC expression in tumour than their respective contro... more <p>Organs with significant higher PDGF-CC expression in tumour than their respective control tissues.</p
<p>Representative images using 6B3 and CD34 antibody in bladder transitional cell carcinoma... more <p>Representative images using 6B3 and CD34 antibody in bladder transitional cell carcinoma (TCC), brain glioblastoma, breast infiltrating duct carcinoma (IDC), kidney renal cell carcinoma (RCC), pancreas and prostate adenocarcinoma (Adca) showed significantly higher expression level of PDGF-CC in comparison to their control tissues (see comments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201089#pone.0201089.t004" target="_blank">Table 4</a>), magnification 200X.</p
PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα ph... more PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα phosphorylation and thereby activates intracellular signalling events essential for development but also in cancer, fibrosis and neuropathologies involving blood-brain barrier (BBB) disruption. In order to elucidate the biological and pathological role(s) of PDGF-CC signalling, we have generated high affinity neutralizing monoclonal antibodies (mAbs) recognizing human PDGF-CC. We determined the complementarity determining regions (CDRs) of the selected clones, and mapped the binding epitope for clone 6B3. Using the monoclonal 6B3, we determined the expression pattern for PDGF-CC in different human primary tumours and control tissues, and explored its ability to neutralize PDGF-CC-induced phosphorylation of PDGFRα. In addition, we showed that PDGF-CC induced disruption of the blood-retinal barrier (BRB) was significantly reduced upon intraperitoneal administration of a chimeric anti-PDGF-CC antibody. In summary, we report on high affinity monoclonal antibodies against PDGF-CC that have therapeutic efficacy in vivo.
It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly... more It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2...
It is demonstrated, using two different perfusion reaction systems, that hybridoma modified by in... more It is demonstrated, using two different perfusion reaction systems, that hybridoma modified by inhibiting their apoptotic response can give improved process performance in terms of cell number and viability in intensive cell culture. Two cell perfusion systems, one using a spin filter and the other an ultrasonic filter, are compared using two cell lines. One cell line is transfected with the bcl-2 gene (TB/C3 bcl-2) which encodes the 'anti-apoptotic' human bcl-2 protein and the other cell line (TB/C3 pEF) with a negative transfection vector. Both reactor systems give similar retention performance for both cell lines. Bcl-2 transfected cells reach higher cell densities than the control cell line, and the percentage of apoptotic cells is clearly lower than with pEF cells. The maximum cell numbers of the bcl-2 cell line are 1.21× 10 7 ml − 1 in the ultrasonic filter culture and 1.58× 10 7 ml − 1 in the spin filter culture, respectively. Using the pEF cell line the maximum cell number reaches 6.0 × 10 6 ml − 1 with ultrasonic retention and 5.9 × 10 6 ml − 1 in the spin filter. The use of ultrasound in this cell retention system has no apparent influence on cell growth, productivity or viability. Selective retention of viable cells is detectable but the effect of removing non-viable cells is negligible.
This report represents an investigation into the nature of apoptosis in hybridoma cultures and it... more This report represents an investigation into the nature of apoptosis in hybridoma cultures and its significance to their utilization in biotechnology. To this end DNA fragmentation and capillary electrophoresis of genomic DNA was studied during the culture of two hybridoma cell lines. This indicated that the phenomenon of apoptosis was always present even under normal culture conditions. Two DNA fragments not associated with the typical DNA fragmentation ladder were identified in the two hybridoma cultures: a previously unreported DNA fragment of about 100 bp and a large fragment which may correspond to one reported in the literature (Walker et al., 1993). The small fragment was identified as soon as the early exponential growth phase of culture, while the large fragment appeared only in the latter part of the growth curve when there was marked DNA fragmentation. In addition we present evidence that aurintricarboxyiic acid, which inhibits apoptosis in neural cells, permits this process in hybridoma cells at levels below 100 PM. This unusual predisposition of hybridoma cultures to undergo apoptosis and their response to inhibitor of apoptosis may have important implications for approaches to the culture of hybridomas and their utilization for monoclonal production.
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Papers by Angelo Perani