Evaluation of an anti-carcinoembryonic monoclonal antibody suitable for immunoscintigraphy

An anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb 6D1.1) was evaluated in vitro and in vivo to determine its suitability as a tracer for immunoscintigraphy of colorectal carcinomas. Determination of mAb affinity for CEA showed a constant of association of 0.63 ± 0.11 x 109 M-1. Binding of technetium-99m (99mTc)-6D1.1, labeled by a direct method, to human cultured lineages was highly specific. Binding to only CEA-positive LS-174T cells resulted in a saturable curve inhibited by pre-incubation with unlabeled mAb. No binding at all was observed for the human lineages MeWo (melanoma) or ZR75-30 (breast carcinoma), neither of them expressing CEA cells. Intravenous injection of 99mTc-6D1.1 into nude mice xenografted with human LS-174T tumors resulted in planar images of excellent quality. Localization of an irrelevant mAb labeled with either 99mTc or iodine-125 was never observed in tumor masses. Biodistribution studies on excised tumoral tissue showed retention of 28.48% of the injected dose per gram of LS-174T tumor. The tumor-to-blood ratio was 3.46. The same analysis performed on the other three human xenografted tumors studied demonstrated that only the CEA-producing HT-29 (colorectal adenocarcinoma) retained 99mTc-6D1.1 while the other two (ZR75-30 and MeWo) did not. These data demonstrate that this mAb is an adequate tool for targeting CEA-expressing tumors in experimental models.

Relationship between the actions of atrial natriuretic peptide (ANP), guanylin and uroguanylin on the isolated kidney

Guanylin and uroguanylin are peptides that bind to and activate guanylate cyclase C and control salt and water transport in many epithelia in vertebrates, mimicking the action of several heat-stable bacteria enterotoxins. In the kidney, both of them have well-documented natriuretic and kaliuretic effects. Since atrial natriuretic peptide (ANP) also has a natriuretic effect mediated by cGMP, experiments were designed in the isolated perfused rat kidney to identify possible synergisms between ANP, guanylin and uroguanylin. Inulin was added to the perfusate and glomerular filtration rate (GFR) was determined at 10-min intervals. Sodium was also determined. Electrolyte dynamics were measured by the clearance formula. Guanylin (0.5 µg/ml, N = 12) or uroguanylin (0.5 µg/ml, N = 9) was added to the system after 30 min of perfusion with ANP (0.1 ng/ml). The data were compared at 30-min intervals to a control (N = 12) perfused with modified Krebs-Hanseleit solution and to experiments using guanylin and uroguanylin at the same dose (0.5 µg/ml). After previous introduction of ANP in the system, guanylin promoted a reduction in fractional sodium transport (%TNa+, P<0.05) (from 78.46 ± 0.86 to 64.62 ± 1.92, 120 min). In contrast, ANP blocked uroguanylin-induced increase in urine flow (from 0.21 ± 0.01 to 0.15 ± 0.007 ml g-1 min-1, 120 min, P<0.05) and the reduction in fractional sodium transport (from 72.04 ± 0.86 to 85.19 ± 1.48, %TNa+, at 120 min of perfusion, P<0.05). Thus, the synergism between ANP + guanylin and the antagonism between ANP + uroguanylin indicate the existence of different subtypes of receptors mediating the renal actions of guanylins.

Interaction between substance P and gastrin-releasing peptide on thyrotropin secretion by rat pituitary in vitro

The effect of substance P (SP) on thyrotropin (TSH) secretion is controversial. In this study we evaluated the effect of SP on TSH secretion by hemipituitaries of 3-month-old Wistar rats in vitro and its interaction with gastrin-releasing peptide (GRP) at equimolar concentrations (1 µM and 10 µM). TSH release was measured under basal conditions and 30 min after incubation in the absence or presence of SP, GRP or both peptides. Pituitary TSH content was also measured in the pituitary homogenate after incubation. SP at both concentrations caused a significant (P<0.05) increase in TSH secretion compared with all other groups, which was approximately 60% (1 µM) and 85% (10 µM) higher than that of the control group (23.3 ± 3.0 ng/ml). GRP at the lower concentration did not produce a statistically significant change in TSH secretion, whereas at the concentration of 10 µM it produced a 50% reduction in TSH. GRP co-incubated with substance P completely blocked the stimulatory effect of SP at both concentrations. Pituitary TSH content decreased in the SP-treated group compared to controls (0.75 ± 0.03 µg/hemipituitary) at the same proportion as the increase in TSH secretion, and this effect was also blocked when GRP and SP were co-incubated. In conclusion, in an in vitro system, SP increased TSH secretion acting directly at the pituitary level and this effect was blocked by GRP, suggesting that GRP is more potent than SP on TSH secretion, and that this inhibitory effect could be the predominant effect in vivo.

Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.

Stabilization of serum antibody responses triggered by initial mucosal contact with the antigen independently of oral tolerance induction

Initial contacts with a T-dependent antigen by mucosal routes may result in oral tolerance, defined as the inhibition of specific antibody formation after subsequent parenteral immunizations with the same antigen. We describe here an additional and permanent consequence of these initial contacts, namely, the blockade of secondary-type responsiveness to subsequent parenteral contacts with the antigen. When repeatedly boosted ip with small doses (3 µg) of ovalbumin (OVA) (or lysozyme), primed B6D2F1 mice showed progressively higher antibody responses. In contrast, mice primed after a single oral exposure to the antigen, although repeatedly boosted, maintained their secondary antibody titers on a level which was inversely proportional to the dose of antigen in the oral pretreatment. This phenomenon also occurred in situations in which oral tolerance was not induced. For example, senile 70-week-old B6D2F1 mice pretreated with a single gavage of 20 mg OVA did not become tolerant, i.e., they formed the same secondary levels of anti-OVA antibodies as non-pretreated mice. However, after 4 weekly challenges with 3 µg OVA ip, orally pretreated mice maintained the same anti-OVA serum levels, whereas the levels of control mice increased sequentially. This "stabilizing" effect of mucosal exposure was dose dependent, occurred with different proteins and was triggered by single or multiple oral or nasal exposures to the antigen.

The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 µM and Emax of 100% (N = 10) or 20 µM and Emax of 92% (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 ± 7.0, 43 ± 3.9 and 78 ± 5.6%) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 µM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 µM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 ± 12%. Glibenclamide (1 or 3 µM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 µM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 µM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 µM, while methylene blue (10 or 30 µM) or ODQ (1 µM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 µM), a VIP receptor antagonist, significantly inhibited (37 ± 7%) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 µM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct activation of voltage and large-conductance Ca+2-activated K+ channels, or indirect modulation of potassium channels induced by VIP receptors and accounts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.

Interactions of laminin with the amyloid ß peptide: Implications for Alzheimer's disease

Extensive neuronal cell loss is observed in Alzheimer's disease. Laminin immunoreactivity colocalizes with senile plaques, the characteristic extracellular histopathological lesions of Alzheimer brain, which consist of the amyloid ß (Aß) peptide polymerized into amyloid fibrils. These lesions have neurotoxic effects and have been proposed to be a main cause of neurodegeneration. In order to understand the pathological significance of the interaction between laminin and amyloid, we investigated the effect of laminin on amyloid structure and toxicity. We found that laminin interacts with the Aß1-40 peptide, blocking fibril formation and even inducing depolymerization of preformed fibrils. Protofilaments known to be intermediate species of Aß fibril formation were also detected as intermediate species of laminin-induced Aß fibril depolymerization. Moreover, laminin-amyloid interactions inhibited the toxic effects on rat primary hippocampal neurons. As a whole, our results indicate a putative anti-amyloidogenic role of laminin which may be of biological and therapeutic interest for controlling amyloidosis, such as those observed in cerebral angiopathy and Alzheimer's disease.

Behavioral and electroencephalographic analysis of seizures induced by intrahippocampal injection of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

In this study, the behavioral and electroencephalographic (EEG) analysis of seizures induced by the intrahippocampal injection in rats of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera, was determined. The first alterations occurred during microinjection of granulitoxin (8 µg) into the dorsal hippocampus and consisted of seizure activity that began in the hippocampus and spread rapidly to the occipital cortex. This activity lasted 20-30 s, and during this period the rats presented immobility. During the first 40-50 min after its administration, three to four other similar short EEG seizure periods occurred and the rats presented the following behavioral alterations: akinesia, facial automatisms, head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements. Within 40-50 min, the status epilepticus was established and lasted 8-12 h. These results are similar to those observed in the acute phase of the pilocarpine model of temporal lobe epilepsy and suggest that granulitoxin may be a useful tool not only to study the sodium channels, but also to develop a new experimental model of status epilepticus.

Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component

Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 10(9) M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography.

The brain decade in debate: VIII. Peptide hormones and behavior: cholecystokinin and prolactin

This article is a transcription of an electronic symposium held on November 28, 2000 in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the peptide field with particular focus on central actions of prolactin and cholecystokinin. The comments in this symposium reflect the diversity of prolactin and cholecystokinin research and demonstrate how the field has matured. Since both peptides play a role in reproductive behaviors, particularly mother-infant interactions, this was the starting point of the discussion. Recent findings on the role of the receptor subtypes as well as interaction with other peptides in this context were also discussed. Another issue discussed was the possible role of these peptides in dopamine-mediated rewarding systems. Both prolactin and cholecystokinin are involved in mechanisms controlling food intake and somatic pain thresholds. The role of peripheral inputs through vagal afferents modulating behavior was stressed. The advent of knockout animals as potential generators of new knowledge in this field was also addressed. Finally, interactions with other neuropeptides and investigation of the role of these peptides in other fields such as immunology were mentioned. Knowledge about the central functions of prolactin and cholecystokinin has shown important advances. The role of these peptides in neurological and psychiatric syndromes such as anorexia, drug abuse and physiological disturbances that lead to a compromised maternal behavior seems relevant.

Serum antigliadin antibody levels as a screening criterion before jejunal biopsy indication for celiac disease in a developing country

The objective of the present study was to determine the efficacy of detection of antigliadin immunoglobulins G and A (IgG and IgA) for the diagnosis of celiac disease in a developing country, since other enteropathies might alter the levels of these antibodies. Three groups were studied: 22 patients with celiac disease (mean age: 30.6 months), 61 patients with other enteropathies (mean age: 43.3 months), and 46 patients without enteropathies (mean age: 96.9 months). Antigliadin IgG and IgA ELISA showed sensitivity of 90.9 and 95.5%, respectively. With the hypothetical values of prevalence ranging from 1:500 to 1:2000 liveborns, the positive predictive value varied from 8.5 to 2.3% for IgG and from 4.8 to 1.1% for IgA. Considering the patients without enteropathies, specificity was 97.8 and 95.7% for IgG and IgA, respectively. In patients with other enteropathies, specificity was 82.0 and 84.1%, respectively. When patients with and without other enteropathies were considered as a whole, specificity was 88.8 and 91.6%, respectively. The specificity of positive IgG or IgA was 93.5% in children without enteropathies and 78.7% in the presence of other enteropathies. The negative predictive value for hypothetical prevalences varying from 1:500 to 1:2000 liveborns was 99.9%. Thus, even in developing countries where the prevalence of non-celiac enteropathies is high, the determination of serum antigliadin antibody levels is a useful screening test prior to the jejunal biopsy in the investigation of intestinal malabsorption.

Nitrergic modulation of vasopressin, oxytocin and atrial natriuretic peptide secretion in response to sodium intake and hypertonic blood volume expansion

The central nervous system plays an important role in the control of renal sodium excretion. We present here a brief review of physiologic regulation of hydromineral balance and discuss recent results from our laboratory that focus on the participation of nitrergic, vasopressinergic, and oxytocinergic systems in the regulation of water and sodium excretion under different salt intake and hypertonic blood volume expansion (BVE) conditions. High sodium intake induced a significant increase in nitric oxide synthase (NOS) activity in the medial basal hypothalamus and neural lobe, while a low sodium diet decreased NOS activity in the neural lobe, suggesting that central NOS is involved in the control of sodium balance. An increase in plasma concentrations in vasopressin (AVP), oxytocin (OT), atrial natriuretic peptide (ANP), and nitrate after hypertonic BVE was also demonstrated. The central inhibition of NOS by L-NAME caused a decrease in plasma AVP and no change in plasma OT or ANP levels after BVE. These data indicate that the increase in AVP release after hypertonic BVE depends on nitric oxide production. In contrast, the pattern of OT secretion was similar to that of ANP secretion, supporting the view that OT is a neuromodulator of ANP secretion during hypertonic BVE. Thus, neurohypophyseal hormones and ANP are secreted under hypertonic BVE in order to correct the changes induced in blood volume and osmolality, and the secretion of AVP in this particular situation depends on NOS activity.

In vitro-induced antibody production in chronic hepatitis C virus infection

The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV) antibody production (IVIAP) in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60% males) with HCV infection (anti-HCV and HCV-RNA positive). Alanine aminotransferase (ALT) was elevated in 89% of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52%) and cirrhosis in 11/52 (21%) patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14% of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540), with 71% sensitivity and 94% specificity for the detection of more advanced disease stages (grades 3 and 4). These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers.

Measurement of Ki-67 antigen in 159 pituitary adenomas using the MIB-1 monoclonal antibody

Pituitary adenomas sometimes show rapid growth and recurrence, and about one third invade the structures surrounding the sella turcica. In an attempt to determine aggressive behavior at an early stage, we used the MIB-1 antibody to identify the Ki-67 antigen. The present study was designed to evaluate pituitary adenomatous tissue in terms of secretion and proliferation and to correlate the Ki-67 index with hormone phenotype and invasive behavior. Material from 159 patients submitted to one or more resections of pituitary adenomas was evaluated. Forty-two non-secretory adenomas and 43 adenomas immunoreactive for growth hormone, 19 for prolactin, 18 for growth hormone and prolactin, 16 for adrenocorticotropic hormone (ACTH), and 21 cases of plurihormonal/gonadotropin adenomas were detected by immunohistochemistry. The MIB-1 antibody was positive in 139 samples and the Ki-67 index ranged from 0.16 to 15.48% (mean = 1.22 ± 2.09%), with no significant difference between genders, age groups, or secretory and non-secretory status. The Ki-67 index was higher in ACTH-secreting adenomas. Invasive pituitary adenomas had a significantly higher Ki-67 index (2.01 ± 3.15%) than macroadenomas with or without supra-sellar extension (1.12 ± 1.87%; P = 0.02). The index was not significantly different in the subgroup of adenomas with invasion of the cavernous sinus compared to groups with other types of invasion. We conclude that tumoral proliferative activity evaluated by the detection of the Ki-67 antigen is significantly higher in invasive than noninvasive adenomas, information which can be useful in therapeutic postoperative management since index cut-off values associated with aggressive behavior can be established.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.