Limits...
Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo.

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells.Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

ABSTRACT
Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

No MeSH data available.


Related in: MedlinePlus

R89A 35 K-Fc expression following hydrodynamic delivery causes significant suppression of zymosan induced peritonitis.Mice were injected with 1 μg 35 K-Fc R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to injection of 100 μg zymosan ip (A). 16 hours after the injection of zymosan recruited cells were recovered by peritoneal lavage and plasma samples harvested for ELISA. Animals receiving R89A 35 K-Fc plasmid expressed 35 K-Fc in the plasma, whereas the protein was not detected in the GFP control animals (B). Leukocytes reovered from the peritoneal cavity were stained with 7/4 and Ly-6G antibodies to detect recruited monocytes and neutrophils (C). Injection of zymosan caused recruitment of both monocytes and neutrophils, but the number of cells recruited was significantly reduced in animals that received R89A 35 K-Fc (D,E). *p < 0.05. Statistical test performed by T-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664965&req=5

f3: R89A 35 K-Fc expression following hydrodynamic delivery causes significant suppression of zymosan induced peritonitis.Mice were injected with 1 μg 35 K-Fc R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to injection of 100 μg zymosan ip (A). 16 hours after the injection of zymosan recruited cells were recovered by peritoneal lavage and plasma samples harvested for ELISA. Animals receiving R89A 35 K-Fc plasmid expressed 35 K-Fc in the plasma, whereas the protein was not detected in the GFP control animals (B). Leukocytes reovered from the peritoneal cavity were stained with 7/4 and Ly-6G antibodies to detect recruited monocytes and neutrophils (C). Injection of zymosan caused recruitment of both monocytes and neutrophils, but the number of cells recruited was significantly reduced in animals that received R89A 35 K-Fc (D,E). *p < 0.05. Statistical test performed by T-test.

Mentions: Prior to evaluating the efficacy of R89A 35 K-Fc in in vivo models of inflammation we first tested whether R89A 35 K-Fc caused any confounding effects on the baseline state of the immune system. We were unable to detect any alteration of spleen:body weight ratio or on circulating leukocyte numbers in the presence of R89A 35 K-Fc expression (Supplementary Figure 2). To test whether the expression of 35 K-Fc following hydrodynamic delivery was functional and sufficient to inhibit CC chemokine mediated inflammation we performed zymosan induced peritonitis experiments in mice that had received R89A 35 K-Fc or a control GFP expression plasmid. To allow full recovery from the general anaesthesia mice were not injected with zymosan until 5 days after hydrodynamic delivery. Mice were injected with 100 ug zymosan to cause a robust recruitment of monocytes and neutrophils to the peritoneum (Fig. 3A). 16 hours after injection with zymosan or saline mice were culled and peritoneal lavage performed and plasma samples prepared. R89 35 K-Fc expression was confirmed in the mice by plasma ELISA and all mice had detectable protein levels with an average of 2.5 μg/ml (Fig. 3B). The cells harvested by lavage were stained to identify monocytes and neutrophils and the recruitment of cells enumerated (Fig. 3C). A small number of saline injected animals were analysed to confirm the expected lack of cells without zymosan injection. We observed a significant reduction (38–40%) in the number of monocytes and neutrophils recruited in animals expressing R89A 35 K-Fc compared to control GFP animals (Fig. 3D,E).


Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo.

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

R89A 35 K-Fc expression following hydrodynamic delivery causes significant suppression of zymosan induced peritonitis.Mice were injected with 1 μg 35 K-Fc R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to injection of 100 μg zymosan ip (A). 16 hours after the injection of zymosan recruited cells were recovered by peritoneal lavage and plasma samples harvested for ELISA. Animals receiving R89A 35 K-Fc plasmid expressed 35 K-Fc in the plasma, whereas the protein was not detected in the GFP control animals (B). Leukocytes reovered from the peritoneal cavity were stained with 7/4 and Ly-6G antibodies to detect recruited monocytes and neutrophils (C). Injection of zymosan caused recruitment of both monocytes and neutrophils, but the number of cells recruited was significantly reduced in animals that received R89A 35 K-Fc (D,E). *p < 0.05. Statistical test performed by T-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664965&req=5

f3: R89A 35 K-Fc expression following hydrodynamic delivery causes significant suppression of zymosan induced peritonitis.Mice were injected with 1 μg 35 K-Fc R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to injection of 100 μg zymosan ip (A). 16 hours after the injection of zymosan recruited cells were recovered by peritoneal lavage and plasma samples harvested for ELISA. Animals receiving R89A 35 K-Fc plasmid expressed 35 K-Fc in the plasma, whereas the protein was not detected in the GFP control animals (B). Leukocytes reovered from the peritoneal cavity were stained with 7/4 and Ly-6G antibodies to detect recruited monocytes and neutrophils (C). Injection of zymosan caused recruitment of both monocytes and neutrophils, but the number of cells recruited was significantly reduced in animals that received R89A 35 K-Fc (D,E). *p < 0.05. Statistical test performed by T-test.
Mentions: Prior to evaluating the efficacy of R89A 35 K-Fc in in vivo models of inflammation we first tested whether R89A 35 K-Fc caused any confounding effects on the baseline state of the immune system. We were unable to detect any alteration of spleen:body weight ratio or on circulating leukocyte numbers in the presence of R89A 35 K-Fc expression (Supplementary Figure 2). To test whether the expression of 35 K-Fc following hydrodynamic delivery was functional and sufficient to inhibit CC chemokine mediated inflammation we performed zymosan induced peritonitis experiments in mice that had received R89A 35 K-Fc or a control GFP expression plasmid. To allow full recovery from the general anaesthesia mice were not injected with zymosan until 5 days after hydrodynamic delivery. Mice were injected with 100 ug zymosan to cause a robust recruitment of monocytes and neutrophils to the peritoneum (Fig. 3A). 16 hours after injection with zymosan or saline mice were culled and peritoneal lavage performed and plasma samples prepared. R89 35 K-Fc expression was confirmed in the mice by plasma ELISA and all mice had detectable protein levels with an average of 2.5 μg/ml (Fig. 3B). The cells harvested by lavage were stained to identify monocytes and neutrophils and the recruitment of cells enumerated (Fig. 3C). A small number of saline injected animals were analysed to confirm the expected lack of cells without zymosan injection. We observed a significant reduction (38–40%) in the number of monocytes and neutrophils recruited in animals expressing R89A 35 K-Fc compared to control GFP animals (Fig. 3D,E).

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells.Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

ABSTRACT
Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

No MeSH data available.


Related in: MedlinePlus