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Repair of CFTR Defects Caused By Cystic Fibrosis MutationsShi, Li 28 November 2013 (has links)
Cystic fibrosis is caused primarily by deletion of Phe508. An exciting discovery was that CFTR’s sister protein, the P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490), could be repaired by a drug-rescue approach. Drug substrates showed specificity, and their mechanism involves direct binding to the transmembrane domains (TMDs) since arginine suppressor mutations were identified in TMDs that mimicked drug-rescue to promote maturation. We tested the possibility of rescuing CFTR processing mutants with a drug-rescue approach. 1) Arginine mutagenesis was performed on TM6, 8, and 12. 2) Correctors were tested for specificity. 3) Truncation mutants were used to map the VX-809 rescue site. Correctors 5a, 5c, and VX-809 were specific for CFTR. VX-809 appeared to specifically rescue CFTR by stabilizing TMD1. Therefore, the TMDs are potential targets to rescue CFTR. Rescue of P-gp and CFTR appeared to occur by different mechanisms since no arginine suppressor mutations were identified in CFTR.
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THE TRANSPORT AND MODULATION OF HIV PROTEASE INHIBITORS INTO THE RAT CENTRAL NERVOUS SYSTEM AND MILKEdwards, Jeffrey Earl 01 January 2004 (has links)
The objective of this dissertation is to study the mechanism by which HIV protease inhibitors enter into the central nervous system (CNS) and breast milk of rats, and what effects MDR modulators have on the distribution and metabolism of HIV protease inhibitors. The transporter P-glycoprotein (P-gp) has been shown to limit the distribution of HIV protease inhibitors into the CNS of rodents. This thesis examined the effects of GF120918, an MDR modulator, on the CNS distribution of amprenavir, an HIV protease inhibitor, in rats. GF120918 significantly increased the unbound CNS concentrations of amprenavir without altering the unbound blood concentrations of amprenavir. The results of these studies show that GF120918 can inhibit P-gp at the blood brain barrier (BBB) to increase the unbound CNS concentration of amprenavir and potentially other HIV protease inhibitors. Many first generation MDR modulators inhibited both P-gp transport and CYP3A metabolism. Therefore, a principal goal of this thesis was to determine if GF120918 could selectively inhibit P-gp transport without inhibiting CYP3A metabolism. Using in vitro (human) and in vivo (rat) studies, GF120918 selectively inhibited P-gp at the BBB without inhibiting CYP3A metabolism. The transporter MRP1 has been shown to both transport HIV protease inhibitors and expressed in the CNS. Studies contained in the thesis have shown that mrp1 is not localized to the BBB of rats, therefore, mrp1 is unlikely to play a significant role in the distribution of HIV protease inhibitors into the CNS of rats. The distribution of nelfinavir, an HIV protease inhibitor, into rat breast milk was studied in the thesis as a first approach in understanding the extent to which HIV protease inhibitors can accumulate into milk. The concentration of nelfinavir in rat milk was approximately half that of plasma. P-gp protein expression was detected in lactating rat mammary tissue. However, GF120918 showed no effect on the distribution of nelfinavir into rat milk suggesting that P-gp does not play a significant role in the distribution of HIV protease inhibitors into milk.
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The effect of different modulators on the transport of rhodamine 123 across rat jejunum using the sweetana-grass diffusion method / C.J. LamprechtLamprecht, Christian Johannes January 2004 (has links)
P-glycoprotein (Pgp), which leads to multidrug resistance in tumour cells,
is an ATP-dependent secretory drug efflux pump. In the intestine, as well as at specific
other epithelial and endothelial sites, P-glycoprotein expression is localised to the apical
membrane, consistent with secretory detoxifying and absorption limitation functions.
The primary function of Pgp is to clear the membrane lipid bilayer of lipophilic drugs.
Results from in vitro studies with human Caco-2 cells provide direct evidence for Pgp
limiting drug absorption. Limitation has non-linear dependence of absorption on
substrate (eg. vinblastine) concentration, increased absorption upon saturation of
secretion and increased absorption upon inhibition of Pgp function, with modulators such
as verapamil. The aim of this study was to investigate the effect of a known Pgp
inhibitor (verapamil) and grapefruit juice components (naringenin, quercetin and
bergamottin) on the transport of Rhodamine 123 across rat jejunum and to compare
these results with those obtained in similar studies done in Caco-2 cells and in rat
intestine (monodirectional). Verapamil, naringenin (442 µM, 662 µM and 884
µM), quercetin (73 µM, 183 µM and 292 µM) and bergamottin (12 µM, 30 µM and 48 µM)
were evaluated as modulators of rhodamine 123 transport across rat jejunum using
Sweetana-Grass diffusion cells. This study was done bidirectionally, with three cells
measuring transport in the apical to basolateral direction (AP / BL) and three cells
measuring transport in the basolateral to apical direction (BL / AP). The rate of transport
was expressed as the apparent permeability coefficient (Papp) and the extent of active
transport was expressed by calculating the ratio of BL/AP to AP/BL.
The BL-AP/AP-BL ratio calculated for Rhodamine 123 with no modulators added was 2.31. The
known modulator verapamil decreased the BL-AP/AP-BL ratio to 1.52. This was
statistically significant and inhibition of active transport was clearly demonstrated. All
modulators inhibited active transport. Only naringenin 884 µM, quercetin 183 µM and
bergamottin 30 µM did not show a statistically significant decrease in the BL-AP/AP-BL
ratio. All three components of grapefruit juice showed inhibition of active
transport and should have an effect on the bioavailability of the substrates of Pgp and
other active transporters. The results obtained in this study are similar to the results
found in Caco-2 cells, which suggests that Sweetana-Grass diffusion method can be
used for diffusion studies. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.
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The effect of selected methoxy flavonoids on the in vitro efflux transport of rhodamine 123 using rat jejunum / Stanley Anthony DoddDodd, Stanley Anthony January 2005 (has links)
Many orally administered drugs must overcome several barriers before
reaching their target site. The first major obstacle to cross is the intestinal epithelium.
Although lipophilic compounds may readily diffuse across the apical plasma membrane,
their subsequent passage across the basolateral membrane and into blood is by no
means guaranteed. Efflux proteins located at the apical membrane, which include P-glycoprotein
(P-gp, MDR1) and Multidrug Resistance-associated Protein (MRP2), may
drive compounds from inside the cell back into the intestinal lumen, preventing their
absorption into the blood. Intestinal P-gp is localised to the villus tip enterocytes, i.e. the
main site of absorption for orally administered compounds and in close proximity to the
lumen. P-gp is therefore ideally positioned to limit the absorption of compounds by
driving efflux back into the lumen. Drugs may also be modified by intracellular phase I
and phase II metabolizing enzymes. This process may not only render the drug
ineffective, but it may also produce metabolites that are themselves substrates for P-gp
and/or MRP2. Drugs that reach the blood are then passed to the liver, where they are
subjected to further metabolism and biliary excretion, often by a similar system of ATP binding
cassette (ABC) transporters and enzymes to that present in the intestine. Thus
a synergistic relationship exists between intestinal drug metabolizing enzymes and
apical efflux transporters, a partnership that proves to be a critical determinant of oral
bioavailability. Aim: The aim of this study was to investigate the effect of selected
methoxy flavonoids (3-methoxyflavone, 5-methoxyflavone, 6-methoxyflavone and 7-
methoxyflavone) on the mean ratio of Rhodamine123 (Rho 123) transport across rat
intestine (jejunum) and to investigate structure activity relationships (SAR) of the
selected flavonoids with reference to inhibition of P-gp. Methods: 3-Methoxyflavone, 5-
methoxyflavone, 6-methoxyflavone and 7-methoxyflavone were evaluated at a
concentration of 10μM and 20μM as modulators of Rho 123 transport across rat
jejunum. The Sweetana-Grass diffusion cells were used to determine the transport of
Rho 123. Each modulator was studied bidirectionally with two cells measuring transport
in the apical to basolateral direction (AP/BL) and two cells measuring transport in the
basolateral to apical direction (BUAP). The rate of transport was expressed as the
apparent permeability coefficient (Papp)and the extent of active transport was expressed
by calculating the ratio of BUAP to AP/BL. Each modulators Papp ratio was then
compared with that of the control. Results: 3-Methoxyflavone decreased the Papp
ratio from 3.34 (control) to 1.66 (10μM) and 1.33 (20μM) and showed statistical
significant differences. 7-Methoxyflavone decreased the Papp ratio to 1.94 (10μM) and
1.55 (20μM) but only showed a statistical significant difference at 10μM. 5-
Methoxyflavone decreased the Papp ratio to 2.41 (10μM) and 1.71 (20μM) and 6-
methoxyflavone decreased the Papp to 3.03 (10μM) and 2.49 (20μM). Both 5- and 6-
methoxyflavone showed no statistical significant differences from the control. The
structure activity relationships with reference to P-gp inhibition clearly indicated that the
C3 and C7 positioning of the methoxy-group on the A ring played a major role in the
inhibition of Rho 123 transport. Conclusion: All the selected modulators showed
inhibition of Rho 123 transport across the jejunum. This should affect the bioavailability
of the substrates of P-gp and other active transporters. In summary, this study describe
the inhibitory interaction of selected flavonoids with P-gp. Structure activity relationships
were identified describing the inhibitory potency of the flavonoids based on methoxy
groups positioning. The inhibitory potency results were 3-methoxyflavone > 7-
methoxyflavone > 5-methoxyflavone> 6-methoxyflavone / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.
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The effect of selected hydroxy flavonoids on the in vitro efflux transport of rhodamine 123 using rat jejunum / S. van HuyssteenVan Huyssteen, Stephanie January 2005 (has links)
Background: Multidrug resistance (MDR) is resistance of cancer cells to multiple
classes of chemotherapeutic drugs that can be structurally unrelated. MDR involves
altered membrane transport that results in a lower cell concentration of cytotoxic drugs
which plays an important role during cancer treatment. P-glycoprotein (Pgp) is localised
at the apical surface of epithelial cell in the intestine and it functions as a biological
barrier by extruding toxic substances and xenobiotics out of cells (Lin, 2003:54). The
ATP-binding-cassette superfamily is a rapidly growing group of membrane transport
proteins and are involved in diverse physiological processes which include antigen
presentation, drug efflux from cancer cells, bacterial nutrient uptake and cystic fibrosis
(Germann, 1996:928; Kerr, 2002:47). A number of drugs have been identified which are
able to reverse the effects of Pgp, multidrug resistance protein (MRPI) and their
associated proteins on multidrug resistance. The first MDR modulators discovered and
studied during clinical trials were associated with definite pharmacological actions, but
the doses required to overcome MDR were associated with the occurrence of
unacceptable side effects. As a consequence, more attention has been given to the
development of modulators with proper potency, selectivity and pharmacokinetic
characteristics that it can be used at a lower dose. Several novel MDR reversing agents
(also known as chemosensitisers) are currently undergoing clinical evaluation for the
treatment of resistant tumours (Teodori et al., 2002:385). Aim: The aim of this study was
to investigate the effect of selected flavonoids (morin, galangin, kaempferol and
quercetin) at two different concentrations (10 μM and 20 μM) on the transport of a known
Pgp substrate, Rhodamine 123 (Rho 123) across rat intestine (jejunum) and to
investigate structure activity relationships (SAR) of the selected flavonoids with
reference to the inhibition of Pgp. Methods: Morin, galangin, kaempferol and quercetin
were evaluated as potential modulators of Rho 123 transport, each at a concentration of
10 μM and 20 μM across rat jejunum using Sweetana-Grass diffusion cells. This study
was done bidirectionally, with two cells measuring transport in the apical to basolateral
direction (AP-BL) and two cells measuring transport in the basolateral to apical direction
(BL-AP). The rate of transport was expressed as the apparent permeability coefficient
(Pap,) and the extent of active transport was expressed by calculating the ratio of BL-AP
to AP-BL. Results: The BL-AP to AP-BL ratio calculated for Rho 123 with no
modulators added was 3.29. Morin decreased the BL-AP to AP-BL ratio to 1.88 at a
concentration of 10 μM and to 1.49 at a concentration of 20 μM. Galangin decreased
the BL-AP to AP-BL ratio to 1.60 at a concentration of 20 μM. These two flavonoids
showed statistically significant results and inhibition of active transport were clearly
demonstrated. However, the other flavonoids inhibited active transport of Rho 123 but
according to statistical analysis, the results were not significantly different. The two
different concentrations (10 μM and 20 μM) indicated that galangin, kaempferol and
quercetin showed practically significant differences according to the effect sizes. Morin,
however, did not show any practically significant differences at the different
concentrations. Regarding .the SAR, it was shown by Boumendjel and co-workers
(2002:512) that the presence of a 5-hydroxyl group and a 3-hydroxyl group as well as
the C2-C3 double bond are required for high potency binding to the nucleotide binding
domain (NBD) of Pgp. All the flavonoids tested had the above-mentioned
characteristics. Conclusion: All the selected flavonoids showed inhibition of active
transport of Rho 123 and should have an effect on the bioavailability of the substrates of
Pgp and other active transporters. This study described the inhibitory interaction of
selected flavonoids on Pgp activity. Practical significant differences between the same
modulator at different concentrations were also observed. Structure activity
relationships were identified describing the inhibitory potency of the flavonoids based on
hydroxyl group positioning / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.
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Blood levels of selective antiretroviral drugs over a period of time, in Sprague-Dawley rats / Michael du PlooyDu Plooy, Michael January 2008 (has links)
Selective antiretroviral! (ARV) drugs are primarily metabolized by cytochrome P450 (CYP) enzymes, characteristically predisposed to variation, and are therefore primarily responsible for ARV pharmacokinetic variability and associated drug interactions. For the majority of ARV drugs, the therapeutic window is narrow and imminent toxicities due to CYP inhibition or sub-therapeutic drug levels as a result of CYP induction is inevitable. Animals provide a metabolism replica to conduct detailed investigations. We endeavored to establish a rat model to screen for variability in metabolism of selective ARV drugs responsible for treatment failure and drug interactions, over time in the liver and serum. Male Sprague-Dawley rats (n = 24) were divided into 6 groups: methylcellulose, 160mg/kg/day (n = 24) (control); efavirenz, 160mg/kg/day (n = 18); ritonavir, 20 mg/kg/day (n = 18); ritonavir, 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day, (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18). Treatment duration varied from one day (single dose), 7 or 21 days. Blood samples were collected after decapitation on days 1, 7 and 21. A sensitive and rapid liquid chromatograph (LC) interfaced to a quadrupoie mass spectrometer (MS) and coupled with electrospray ionization (ESI) method was employed for the blood sample determinations. One single injection was required to simultaneously quantify efavirenz, lopinavir and ritonavir within the linear concentration range of 78 - 5000 ng/ml. Efavirenz blood levels increased statistically significantly (p < 0.05) from day 1 to day 21 with distinct steady state achievement prior to day 7. The levels of ritonavir increased statistically significantly (p < 0.05) from day 7 to 21 when administered alone and statistically significantly (p < 0.01) from day 1 to 21 when administered as the ritonavir/lopinavir combination. The levels of lopinavir also increased statistically significantly (p<0.01) from day 1 and 21 in the ritonavir/lopinavir combination. However, the inclusion of a P-glycoprotein inhibitor, verapamil, increased both the ritonavir (administered alone) and lopinavir blood levels significantly (p < 0.05) at day 1. The ritonavir levels were also significantly increased on day 21 (p < 0.05). When verapamil was added to the ritonavir/lopinavir combination the levels of ritonavir increased statistically significantly (p < 0.01) from day 1 to 21. A rat model can be used to detect changes in metabolism over time as measured by blood levels. The influence of drug interactions, such as verapamil, on ARV drug metabolism can be investigated by this model. These results will be substantiated by PCR liver results in the future. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2009.
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The Role of Corticosteroids in Nitrogen Excretion of the Gulf Toadfish (Opsanus beta)Rodela, Tamara 03 May 2011 (has links)
In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity.
Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea.
This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.
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Examining the integrity of the blood-brain barrier (BBB) and the use of lysophosphatidic acid (LPA) to modulate the barrier propertiesOn, Ngoc H. 03 1900 (has links)
INTRODUCTION: The blood brain barrier (BBB), formed by the brain capillary endothelial cells separating the blood from the brain. Furthermore, the brain endothelial cells also express numerous transporter systems which help regulate and maintain the brain microenvironment. The protective function of the BBB and their transporter systems under pathological disease states, including brain tumor, can be an obstacle for the entry of therapeutic agents to the brain.
OBJECTIVES: The current study set out to characterize brain tumor-induced alterations of the BBB of a mouse brain tumor model. Studies were performed to address changes in BBB permeability to P-gp dependent solutes using Rhodamine (R800). Furthermore, the use of lysophosphatidic acid (LPA) to modulate BBB permeability was also examined in healthy mice and tumor-bearing mice.
METHODS: Tumors were induced by injecting Lewis Lung carcinoma (3LL) cells into the right hemisphere of female Balb/c mice. Changes in BBB permeability were assessed at various stages of tumor development, using both gadolinium contrast-enhanced agent (Gad) and 3H-mannitol. Functional activity of P-gp in the BBB was examined in adult mice following i.v. injection of R800 in the presence and absence of GF120918 (a P-gp inhibitor). Alterations in BBB permeability were characterized in healthy and tumor-bearing mice using a small (Gad) and large (IRdye800cw PEG) vascular permeability agent as well as R800 (changes in P-gp mediated permeability).
RESULTS: Median mouse survival following 3LL injection was 17 days. The BBB was largely intact during tumor development with disruptions observed at the later stages of tumor development as indicated by Gad permeability. By inhibiting the function of P-gp with GF120918, the distribution of R800 in the brain increased by 4-fold. The enhancement effect of LPA on BBB permeability occurs within 3-6 minutes of injection with the barrier being restored back to its normal function within 20 minutes. Furthermore, an increased in brain penetration of IRdye800ce PEG and R800 were observed following LPA injection in both healthy and tumo-bearing mice.
CONCLUSION: These studies provide the initial proof of concept for the use of BBB modulators including LPA and GF120918 to enhance drug delivery to the brain and the tumor sites.
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Blood levels of selective antiretroviral drugs over a period of time, in Sprague-Dawley rats / Michael du PlooyDu Plooy, Michael January 2008 (has links)
Selective antiretroviral! (ARV) drugs are primarily metabolized by cytochrome P450 (CYP) enzymes, characteristically predisposed to variation, and are therefore primarily responsible for ARV pharmacokinetic variability and associated drug interactions. For the majority of ARV drugs, the therapeutic window is narrow and imminent toxicities due to CYP inhibition or sub-therapeutic drug levels as a result of CYP induction is inevitable. Animals provide a metabolism replica to conduct detailed investigations. We endeavored to establish a rat model to screen for variability in metabolism of selective ARV drugs responsible for treatment failure and drug interactions, over time in the liver and serum. Male Sprague-Dawley rats (n = 24) were divided into 6 groups: methylcellulose, 160mg/kg/day (n = 24) (control); efavirenz, 160mg/kg/day (n = 18); ritonavir, 20 mg/kg/day (n = 18); ritonavir, 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day, (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18). Treatment duration varied from one day (single dose), 7 or 21 days. Blood samples were collected after decapitation on days 1, 7 and 21. A sensitive and rapid liquid chromatograph (LC) interfaced to a quadrupoie mass spectrometer (MS) and coupled with electrospray ionization (ESI) method was employed for the blood sample determinations. One single injection was required to simultaneously quantify efavirenz, lopinavir and ritonavir within the linear concentration range of 78 - 5000 ng/ml. Efavirenz blood levels increased statistically significantly (p < 0.05) from day 1 to day 21 with distinct steady state achievement prior to day 7. The levels of ritonavir increased statistically significantly (p < 0.05) from day 7 to 21 when administered alone and statistically significantly (p < 0.01) from day 1 to 21 when administered as the ritonavir/lopinavir combination. The levels of lopinavir also increased statistically significantly (p<0.01) from day 1 and 21 in the ritonavir/lopinavir combination. However, the inclusion of a P-glycoprotein inhibitor, verapamil, increased both the ritonavir (administered alone) and lopinavir blood levels significantly (p < 0.05) at day 1. The ritonavir levels were also significantly increased on day 21 (p < 0.05). When verapamil was added to the ritonavir/lopinavir combination the levels of ritonavir increased statistically significantly (p < 0.01) from day 1 to 21. A rat model can be used to detect changes in metabolism over time as measured by blood levels. The influence of drug interactions, such as verapamil, on ARV drug metabolism can be investigated by this model. These results will be substantiated by PCR liver results in the future. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2009.
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Identification of anti-beta₂ glycoprotein I auto-antibody regulated gene targets in the primary antiphospholipid syndrome using gene microarray analysisHamid, Colleen G. January 2007 (has links)
Anti-Beta2-Glycoprotein I antibodies (anti-b2GPI) are strongly associated with thrombosis in patients with primary antiphospholipid syndrome (PAPS). Anti-b2GPI activate endothelial cells (EC) resulting in a pro-thrombotic and pro-inflammatory phenotype. In order to characterise EC gene regulation in response to anti-b2GPI, early global gene expression was assessed in human umbilical vein endothelial cells (HUVEC) in response to affinity purified anti-b2GPI. Sera were collected from patients with PAPS and IgG was purified using HiTrap Protein G Sepharose columns. Polyclonal anti-b2GPI were prepared by passing patient IgG through NHS activated sepharose coupled to human b2GPI. Anti-b2GPI preparations were characterized by confirming their b2GPI co-factor dependence, binding to b2GPI and ability to induce leukocyte adhesion molecule expression and IL-8 production in vitro. Two microarray experiments tested differential global gene expression in 6 individual HUVEC donors in response to 5 different PAPS polyclonal anti-b2GPI (50 mg/ml) compared to 5 normal control IgG (50 mg/ml) after 4 hours incubation . Total HUVEC RNA was extracted and cRNA was prepared and hybridised to Affymetrix HG-133A (Exp.1) and HG-133A_2 (Exp.2) gene chips. Data were analyzed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. Significant change in gene expression was defined as greater than two fold increase or decrease in expression (p<0.05). Novel genes not previously associated with PAPS were induced including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1 and growth factors, CSF2, CSF3, IL-6, IL1b and FGF18. Downregulated genes were transcription factors/signaling molecules including ID2. Microarray results were confirmed for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C) using quantitative real-time RT-PCR analysis. This study revealed a complex anti-b2GPI-regulated gene expression profile in HUVEC in vitro. The novel chemokines and pro-inflammatory cytokines identified in this study may contribute to the vasculopathy associated with PAPS.
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