Standardization of the homogeneous mobility shift assay protocol for evaluation of anti-infliximab antibodies. Application of the method to Crohn's disease patients treated with infliximab.

BACKGROUND: The availability of a quantitative method to measure anti-infliximab (IFX) antibodies (ATI) would facilitate the implementation of therapeutic drug monitoring in clinical decision-making. Our aim was to standardize the homogeneous mobility shift assay (HMSA) used in the measure of ATI levels. METHODS: In this prospective longitudinal multicenter study, 50 IFX-treated Crohn's disease (CD) patients were followed up for 54weeks. During this period 360 human serum samples were analysed. Monomeric ATI levels were measured by a quantitative HMSA-method using an anti-IFX calibrator. IFX trough levels measured by ELISA were correlated with ATI levels. RESULTS: Using HMSA and a pure anti-idiotypic monoclonal antibody specific for IFX (anti-IFX calibrator), we measured the levels of monomeric ATI generated in Crohn's disease patients treated with IFX. Anti-IFX calibrator allowed to quantify monomeric antibodies against IFX with a low limit of quantification (3nM). The threshold level of ATI in order to classify the immunogenicity of the patients was 10nM. We observed that 24% (12/50) of IFX-treated patients developed ATI (>10nM) during the observation period (54weeks). Serum concentration of ATI higher than 10nM dramatically increased the probability (OR=51.1; 95% CI: 20.4-128.0; p<0.0001) of presenting low levels of IFX (⩽1.5nM) in serum, as observed in some CD patients treated with standard doses of the drug. CONCLUSIONS: The HMSA-method described here allows an accurate quantification of ATI concentration in international units (IU) and therefore it could be useful in the study of the relationship between ATI concentration, infliximab level and the clinical response to the drug.

In vitro studies of water-stable cationic carbosilane dendrimers as delivery vehicles for gene therapy against HIV and hepatocarcinoma.

Here we present a synthetic procedure for water-stable carbosilane dendrimers containing ammonium groups at the periphery of type Gn-{[Si(CH2)3N+(Me)(Et)CH2CH2N+Me3]x (CF3SO3 -)y} which have been used as non-viral vectors for transfecting different types of nucleic acids against two different medical problems, HIV and hepatocarcinoma. These systems have shown to be non-toxic in both PBMC and HepG2 cell lines under the experimental conditions and are able to form nanoconjugates with nucleic acids perfectly stable over time and in a wide range of pH values, which leads to the conclusion that the interaction between dendrimer and nucleic acid is very strong. In addition, a high degree of transfection using these nanoconjugates has been observed, ranging from 70-90% depending on the generation and in the particular case of PBMC transfection with anti-HIV oligonucleotides. However, besides of the good properties shown by the dendrimers here prepared as transfecting agents, only moderate effect was observed in functional experiments for hepatocarcinoma, as a result of the strong interaction between dendrimer and nucleic acid. Nevertheless, it is important to mention that an IRS-4 knock-down of 40% in HepG2 achieves an analogous degree of cell sensitization to cancer treatment, which may represent a major advance in the hepatocarcinoma treatment when appropriate dendrimers as transfection agents are used.

Association of adenylate cyclase with an actin-like protein in the human myometrium.

Regulation of muscle contraction by second messengers such as cAMP and regulation of the adenylate cyclase enzyme by the cytoskeleton have been previously described. However, the physical association of both effector and structural elements is still unknown. In this context, we have co-purified a human myometrial adenylate cyclase with an actin-like protein in a two-step purification protocol. The adenylate cyclase catalytic unit was solubilized with Lubrol-PX, submitted to anionic exchange chromatography and purified about 7-fold. The eluate was applied to a forskolin-agarose column obtaining an adenylate cyclase extract enriched 257-fold (enzymatic activity of 1390 pmol/30 min per mg protein) that co-eluted with a 74.6-kDa protein that possessed the 18-27 amino-acid fragment from the N-terminal region of human actin. Under non-reducing conditions, the apparent molecular weight of this protein decreased to 54 kDa, which has been previously described for arthrin. These results provide the first demonstration of the physical association of human myometrial adenylate cyclase with a cytoskeleton-related protein, supporting the hypothesis that adenylate cyclase is regulated by mechanical stimuli.

Thiopurine methyltransferase activity in a Spanish population sample: decrease of enzymatic activity in multiple sclerosis patients.

The present study was performed in order to obtain the thiopurine methyltransferase (TPMT) activity frequency distribution histogram in a Spanish population. A total of 3640 Spanish clinical laboratory samples were evaluated, which included 1249 patients with Crohn's disease, 589 with ulcerative colitis, 348 with multiple sclerosis (MS), 487 with several autoimmune diseases different from the above-mentioned diseases and 967 a donor group. We have measured the TPMT activity in red blood cells (RBCs) by a radiochemical method, using S-adenosyl-L-[methyl-3H]methionine as methyl donor. The different groups present in their entirety a normal distribution histogram and a wide range of TPMT activity from 0 to 41 U/ml RBCs. The differences found between the Spanish population TPMT activity frequency distribution histogram and the pattern previously described in a North American population were not due to azathioprine treatment or gender. The effect of autoimmune diseases on TPMT activity was evaluated: the enzymatic activity was similar in the donor group (19.9 +/- 6.3 U/ml RBCs) and in the patients with Crohn's disease (20.0 +/- 5.8 U/ml RBCs) and ulcerative colitis (19.7 +/- 6.1 U/ml RBCs); however, it decreased significantly (p<0.0001) in MS patients (17.1 +/- 6.1 U/ml RBCs) with respect to the donor group. In conclusion, our results show that the Spanish population TPMT distribution is closer to that of the Jewish population of Israel than to North American populations, and that in MS the enzymatic activity of TPMT decreases significantly. This observation may take into account the usage of azathioprine as therapeutic agent in Spanish MS patients.

Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: comparison with the radiochemical method.

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.

Vasoactive intestinal peptide (VIP) stimulates rat prostatic epithelial cell proliferation.

BACKGROUND: Androgens play a major role in supporting normal growth and functional maintenance in the prostate. However, this gland contains an array of neuroendocrine peptides that can play a regulatory role in its physiopathology. Among these peptides, one of the best studied is vasoactive intestinal peptide (VIP), which is abundant in autonomic nerves surrounding both human and rat prostatic acini. This neuropeptide may act through interaction with two types of high-affinity receptors, named VPAC(1) and VPAC(2) receptors. Another regulatory peptide, the pituitary adenylate cyclase-activating peptide (PACAP), interacts with these receptors with the same affinity as VIP, but binds with higher affinity to PAC(1) receptors. Human prostate tumors and rat prostate show a major presence of VPAC(1) receptors, whereas various findings suggest a role for VIP in prostatic development. Here we studied the effects of VIP on the proliferation of rat prostatic epithelial cells in culture. METHODS: We studied the [(3)H]-thymidine uptake by rat prostatic epithelial cells in culture, characterized previously by using biomarkers such as cytokeratin and vimentin. In these cells we tested the effect of VIP and PACAP-27 on two different signaling pathways, the cyclic AMP (cAMP) and the inositol phosphate (IPs). RESULTS: The rat prostatic cells in culture were cytokeratin (5,6,8) and vimentin positive, indicating that the culture was predominantly epithelial. The proliferation curves showed that the cells followed different states of growth: a quiescent, an exponential proliferative, and a steady state. Cyclic AMP production, but not inositol phosphate production, was increased in the presence of VIP and PACAP-27, which suggests the expression of VPAC(1) and/or VPAC(2) receptors primarily. VIP significantly increased prostatic cell proliferation in a bimodal manner, as shown for dibutyryl cyclic AMP (dbcAMP), which suggests that the effect of VIP upon prostatic proliferation is cAMP-dependent. CONCLUSIONS: Here, we demonstrate that VIP increased [(3)H]thymidine uptake by rat prostatic epithelial cells in culture, conceivably by the activation of the adenylate cyclase.

Expression of vasoactive intestinal peptide (VIP) receptors in human uterus.

We show the existence of functional vasoactive intestinal peptide (VIP) receptors in normal human female genital tract (endometrium, myometrium, ovary and Fallopian tube) as well as in leiomyoma (a frequent uterine pathology). The correlation between VIP binding and stimulation of adenylyl cyclase activity for all studied tissues was linear (r = 0.86) suggesting the expression of VIP receptors throughout the human female genital tract. Immunodetection of VIP receptor subtypes gave different molecular weights for VPAC(1) (47 kDa primarily) and VPAC(2) (65 kDa), which may be due to different glycosylation extents. In conclusion, this study demonstrates the expression of both subtypes of VIP receptors and their functionality in human female genital tract, suggesting that this neuropeptide could play an important physiological and pathophysiological role at this level.

Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma.

The mechanisms responsible for the growth of uterine leiomyoma (a frequent cause of infertility in women) are largely unknown. Some data supports that cAMP plays a role in the growth of uterine cells but there are no reports on the status of the cAMP producing system in this human benign neoplasia. In this study, biopsies from leiomyoma and the adjacent myometrium were taken from menstruating women subjected to total hysterectomy for leiomyoma. Adenylate cyclase activity was determined by a protein-binding method, and the expression of alpha(s), alphai1/2, alphai3 and alphai0) G-protein subunits was analysed by immunoblot. The leiomyoma samples exhibited a decreased expression of as and ai1/2 with respect to the adjacent myometrial tissue. No differences were observed in alphai3 and alphaio protein expression. The basal adenylate cyclase activity as well as the efficacy (as assessed by the maximal stimulation levels) of either forskolin or, to a lesser extent, Gpp[NH]p on stimulation the enzyme activity was significantly lower in leiomyoma than in myometrium, whereas the potency (as assessed by the ED50 values) of these two agents did not vary. Present data indicate that the human leiomyoma is associated with low levels of cAMP. It is conceivable that the loss of sensitivity of adenylate cyclase to endogenous regulatory molecules could be related to the pathogenesis of human leiomyomas given that cAMP inhibits the MAP-kinase cascade in uterine tissues.

Expression, pharmacological, and functional evidence for PACAP/VIP receptors in human lung.

Pituitary adenylate cyclase-activating peptide (PACAP) type 1 (PAC(1)) and common PACAP/vasoactive intestinal peptide (VIP) type 1 and 2 (VPAC(1) and VPAC(2), respectively) receptors were detected in the human lung by RT-PCR. The proteins were identified by immunoblotting at 72, 67, and 68 kDa, respectively. One class of PACAP receptors was defined from (125)I-labeled PACAP-27 binding experiments (dissociation constant = 5.2 nM; maximum binding capacity = 5.2 pmol/mg protein) with a specificity: PACAP-27 approximately VIP > helodermin approximately peptide histidine-methionine (PHM) >> secretin. Two classes of VIP receptors were established with (125)I-VIP (dissociation constants of 5.4 and 197 nM) with a specificity: VIP approximately helodermin approximately PACAP-27 >> PHM >> secretin. PACAP-27 and VIP were equipotent on adenylyl cyclase stimulation (EC(50) = 1.6 nM), whereas other peptides showed lower potency (helodermin > PHM >> secretin). PACAP/VIP antagonists supported that PACAP-27 acts in the human lung through either specific receptors or common PACAP/VIP receptors. The present results are the first demonstration of the presence of PAC(1) receptors and extend our knowledge of common PACAP/VIP receptors in the human lung.

Effects of chronic ethanol ingestion on the vasoactive intestinal peptide receptor-effector system from rat seminal vesicle membranes.

We studied the modifications of the vasoactive intestinal peptide (VIP) receptor/effector system from the rat seminal vesicle after chronic ethanol ingestion. Ethanol treatment resulted in a decreased height of the secretory epithelium of seminal vesicle as well as in a weight loss of this gland. These morphological changes were accompanied by an increase of immunoreactive vasoactive intestinal peptide (VIP) levels and a decrease of the stimulatory effect of VIP adenylate cyclase activity in the seminal vesicle. The loss of sensitivity of the enzyme to VIP was conceivably related to a decrease in the affinity of VIP receptors rather than to a decrease in their number. The changes in the affinity of the VIP receptors were accompanied with a lower sensitivity of VIP binding to GTP, which suggest an uncoupling between the receptor and the transductor molecules. However, chronic exposure to ethanol did not modify either the levels of G-protein subunits (alpha(s) and alpha(i1/2)) or the GTPase activity from seminal vesicle membranes. Moreover, ethanol feeding did not affect adenylate cyclase activity stimulated by forskolin or by Gpp(NH)p. Thus, ethanol-induced changes in the sensitivity of adenylate cyclase to VIP appear to be attributed to an alteration in the VIP-receptor/G-protein interphase rather than in the G-protein/adenylate cyclase connection.

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate.

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.

Cyclosporin A induces apoptosis in rat hepatocytes in culture.

Cyclosporin A (CsA) at concentrations up to 1 microM induced apoptosis in a dose-dependent manner in cultured rat hepatocytes for 48 h in the presence of insulin and epidermal growth factor (EGF). The effect of CsA was evidenced by the DNA fragmentation pattern constituted by fragments of multiples of 180-200 base pairs, which is a characteristic of programmed cell death. The metabolic activity did not change significantly in the presence of 0.1 microM CsA and diminished to 49% of control in the presence of 1 microM CsA. Changes in the metabolic activity were correlated with a decrease in both [methyl-3H]thymidine uptake and DNA content, which reflects a decrease in the cell number. The treatment of cells with CsA (1 microM) decreased the metabolic activity/DNA content ratio by 24% with respect to dimethyl sulphoxide (DMSO) control, which also suggests, under these conditions, that the necrosis achieved is at most only 24%. In addition, the changes observed (apoptotic process, arrest of the cell cycle and apparition of a necrotic process) were correlated with an increase in the high-affinity guanosine triphosphatase (GTPase) enzymes.

G-protein regulation of adenylate cyclase activity in rat prostatic membranes after chronic ethanol ingestion.

BACKGROUND: The possibility that long-term ethanol ingestion might alter either vasoactive intestinal peptide (VIP) content, VIP binding to membrane receptors, G-protein levels or adenylate cyclase activity in rat prostate was tested, as ethanol produces serious alterations in the hypothalamic-pituitary-gonadal axis and several modifications on different elements on signal transduction pathways in other systems. METHODS: Prostatic membranes from control and ethanol-treated (for 4 weeks) rats were used to study adenylate cyclase stimulation as well as for the immunodetection of stimulatory (alpha(s)) and inhibitory (alpha(i)1-2) G-protein subunits. Studies on VIP binding and cross-linking to receptors were performed using [125I]VIP. Prostatic VIP content was estimated by radioimmunoassay. GTPase activity was quantified by measuring the amount of 32Pi released from [gamma-32P]GTP. RESULTS: Chronic ethanol ingestion resulted in an increased presence of VIP in the rat prostate without any change on the VIP receptor/effector system in this gland. By contrast, the basal adenylate cyclase activity as well as the dose-dependent stimulation of this enzyme by either the nonhydrolyzable GTP analogue Gpp(NH)p or the beta-adrenergic agonist isoproterenol were enhanced in prostatic membranes after ethanol intake. Moreover, an increase in the content of G-protein subunits (alpha(S) and alpha(i)1-2) was observed without any change in GTPase activity in this condition. These modifications were accompanied by a significant decrease in rat prostate weight and, consequently, the height of the secretory epithelium in this gland. CONCLUSIONS: Considering the role of VIP in the mechanisms of secretion and cell proliferation in the prostate, the observed increases in the prostatic content of VIP and G-protein subunits make conceivable that VIP and cAMP signal transduction could be involved in the atrophy of the rat prostate and in the alterations in the composition of seminal fluid that appear in the alcoholic syndrome.

Inhibitory effect of cyclosporin A peptide on rat hepatocytes proliferation induced by mitogens.

Treatment of cultured rat hepatocytes with cyclosporin A (0.01-1 microM) for 24, 48, or 72 h in the presence of insulin and epidermal growth factor induced an inhibition on cell proliferation in a time- and concentration-dependent manner, with an IC50 = 0.05 microM CsA corresponding to 48-h treatment. The inhibitory effect of CsA at < or = 0.1 microM doses for 48 h on [3H]thymidine uptake was reversed after withdrawal of the drug and subsequent addition of insulin plus EGF or serum; however, at 1 microM CsA the effect was irreversible and numerous bright small vesicles were observed. The molecular mechanism involved in CsA action in hepatocytes seems to be independent on cAMP and pertussis-toxin sensitive G proteins.

Ontogenic development of the adenylyl cyclase enzyme and the alpha s, alpha i1 and alpha i2 G-protein regulatory subunits from rat prostate.

The expression of alpha s, alpha i1 and alpha i2 G-protein subunits measured by immunoblot increased in the rat prostate during sexual maturation, supporting their involvement in proliferation/differentiation. Northern blotting gave transcripts of 1.8 and 4 kb for alpha s, 1.4 and 4.5 kb (mainly) for alpha i1, and 2.4 kb for alpha i2 with levels suggesting a differential regulation (at transcription or post-transcription for alpha s, transcription for alpha i1, and translation for alpha i2). The stimulatory effects of forskolin, vasoactive intestinal peptide (VIP) and isoproterenol on adenylyl cyclase activity increased between 0.5-3 mo, remained constant up to 12 mo and decreased thereafter, conceivably following the expression of VIP and beta-adrenergic receptors. However, G-protein activation of adenylyl cyclase (by GTP and Gpp[NH]p) was maximal at 0.5 mo and then decreased as it occurred with toxin-catalyzed ADP-ribose incorporation to alpha subunits suggesting that other factors are also involved in the regulation of G-protein activity during rat prostatic development.

Characterization of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptors in human benign hyperplastic prostate.

Vasoactive intestinal peptide (VIP) is an important member of the group of neuropeptides that appears to be involved in the regulation of prostatic growth and function. Here we studied VIP receptors in membranes from human benign hyperplastic prostate. Accordingly to observations in rat prostatic membranes, [125I]VIP binding to human prostatic membranes suggested two classes of binding sites with high Kd = 0.22 nM) and low (Kd = 37.7 nM) affinities. VIP bound in human and rat membrane preparations to a common VIP/pituitary adenylate cyclase-activating peptide (PACAP) receptor, as VIP, PACAP-27, and PACAP-38 were equipotent for competition of [125I]VIP binding. A PACAP-preferring receptor appears to be expressed in human prostate, since [125I]PACAP binding was displaced with more potency by PACAP than by VIP, and a messenger RNA corresponding to type I PACAP receptor was found. Cross-linking experiments suggested a VIP receptor of about 71 kDa in human and 52 kDa in rat prostates. The binding of [125I]VIP to membranes and the labeling of the bands observed after electrophoresis were competitively inhibited by GTP, suggesting the coupling of VIP receptors to a G protein. Moreover, after solubilization and cross-linking, we observed a 120-kDa band that corresponded to the VIP receptor-alpha s association. VIP stimulated adenylyl cyclase activity in a dose-dependent manner, but the potency and/or the efficacy of VIP were lower in all human preparations studied than in rat prostatic membranes. In conclusion, this study clearly demonstrates the expression of VIP/PACAP common receptors associated with alpha s protein in human prostate and suggests that these neuropeptides could play an important and complex role in the physiology and pathophysiology of this human gland.

Characterization of adenylyl cyclase stimulated by VIP in rat and mouse peritoneal macrophage membranes.

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in rat and mouse peritoneal macrophage membranes. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 microM GTP. Other agents like GTP, Gpp(NH)p, GTP-gamma-S, sodium fluoride, and forskolin, at a concentration of 0.1 mM, increased the basal activity of enzyme by 3.1, 5.7, 4.7, 3.6, and 7.8-fold, respectively. The stimulation of adenylyl cyclase by VIP was time, temperature, and membrane concentration dependent. Half-maximal enzyme activation (ED50) was very similar in rat and mouse peritoneal macrophage membranes (1.5 +/- 0.1 nM and 1.0 +/- 0.1 nM, respectively). However, VIP showed more efficacy in mouse macrophages membranes (about 3.1-fold basal values) than that in rat macrophage membranes (about 2.5-fold basal values). The relative potency of several peptides upon stimulation of adenylyl cyclase activity showed the following potency in both species: VIP = PACAP38 = PACAP27 > helodermin > PHI > secretin. On the other hand, a M(r)-45 kDa alpha s subunit of Gs protein was demonstrated by both ADP-ribosylation and immunoblot in mouse and rat peritoneal macrophage membranes. The present results, together other previous, strongly suggest that VIP play an important role in the regulation of macrophage function.

Protein kinase C isozymes in prostatic epithelial cells from normal, diabetic and insulin-treated diabetic rats.

1. Immunoblot experiments in rat prostatic epithelium using a non-selective antibody against protein kinase C (PKC) allowed to detect three PKC subspecies of 87.5, 55.5 and 34.6 kDa that showed higher, similar and lower immunoreactivity in the membrane than in the cytosolic compartment, respectively. 2. Specific monoclonal antisera revealed that the PKC-gamma isozyme is not expressed in the rat prostatic epithelium, whereas the PKC-beta isozyme was noted only in the cytosolic fraction showing an apparent molecular weight of 75.5 kDa. 3. Induction of diabetes by streptozotocin led to modifications in the expression of PKC isozymes so that the immunoreactivities of the 87.5- and 55.5-kDa PKC forms decreased in both cytosolic and membrane subcellular fractions to different extents. 4. The most important decrease was that of the 55.5-kDa PKC form in cytosol that returned to control values by insulin therapy, whereas PKC-beta suffered also some decrease in diabetes and increased again with insulin treatment.