Natural killer clones derived from fetal (25 wk) blood. Probing the human T cell receptor with WT31 monoclonal antibody.

We have conducted a phenotypic and functional analysis of 19 cloned cell lines generated after allogeneic stimulation of circulating lymphocytes from a normal human fetus aged 25 wk. Using a limited series of mAbs (Anti-T3, WT31, T4, T8, and NKH1A), cloned cells were found to fall in three groups. Three clones have a conventional "inducer" phenotype. Three clones have a phenotype (T3+, WT31+, T8+, and NKH1A+) similar to that of certain NK active mature T lymphocytes present in adult peripheral blood. In contrast, 13 cell lines display surface characteristics that have not been described previously. Indeed, they express T3 proteins but not the WT31 determinant. In light of previous studies, these results show that WT31 mAb is a unique reagent directed at an invariant epitope of the human T cell receptor that is not present on all circulating T3+ fetal lymphocytes. Functionally the T3+, WT31+, and NKH1A+ clones were found to kill immunizing LAZ 388 cells, as well as K562, while T3+, WT31- and NKH1A+ clones display NK-like function exclusively. Moreover, only WT31+ lymphocytes present in the cell line used for cloning experiments have the capacity to recognize alloantigen-bearing cells. Together, these data suggest that expression of WT31 may be necessary for recognition of alloantigens, while NK reactions mediated by T3+ lymphocytes are WT31-independent.

Peptide immunogen mimicry of a protein-specific structural epitope on human choriogonadotropin.

It is a challenge to construct synthetic immunogens that elicit antibodies (Abs) both directed to conformational epitopes and specific for a complex protein like human choriogonadotropin (hCG). A monoclonal antibody specific for hCG bound to regions around Lys45 of the alpha subunit (hCG alpha) and Asp112 of the beta subunit (hCG beta). A peptide comprising residues 46 to 55 of hCG alpha and residues 106 to 116 of hCG beta elicited Abs in rabbits that were directed to a discontinuous epitope and were specific for hCG. These Abs inhibited the binding of hCG to its receptor. Thus, a synthetic immunogen can mimic a conformational-specific epitope and can be useful for vaccine development.

Catecholamine metabolism in neuroblastoma.

Previous studies indicating the importance of catecholamine metabolism in neuroblastoma were briefly reviewed. Metabolic pathways were presented showing how the major urinary metabolites 3-methoxy-4-hydroxymandelic acid (VMA) and 3-methoxy-4-hydroxy-phenylacetic acid (HVA) are formed from norepinephrine and from dopamine plus 3,4-dihydroxyphenylalanine (DOPA), respectively. For 289 neuroblastoma patients at the time of diagnosis, the urinary excretion of VMA was significantly elevated in 75%, and HVA was elevated in 80%. Periodic assay of these metabolites during the course of the disease revealed that the excretion trends were of prognostic value with 80-90% reliability. By contrast, when the excretion in only the initial urine specimens was considered, the survival rate was the same for patients with normal, and with significantly elevated, excretion. Review of the results of tracer studies aimed at elucidating the in vivo metabolic origins of the urinary metabolites suggested that a) in neuroblastoma, the catecholamines were largely inactivated by intracellular metabolism in the tumor cells; b) there was excess production and excretion of the norepinephrine precursors, DOPA and dopamine; and c) in the tumors of most neuroblastoma patients, the initial enzyme in catecholamine synthesis, tyrosine hydroxylase, had an activity comparable with that in normal adrenal glands. The importance of the metabolism of catecholamines in patients with neuroblastoma was stressed: a) The excretion of elevated levels of urinary catecholamine metabolites were useful in diagnosis and in following the course of the disease, and b) study of the catecholamine metabolism in these patients permitted examination of possible relationships between the activity of the enzymes involved in catecholamine synthesis and the malignancy of this tumor.

Identification and measurement of calcitonin precursors in serum of patients with malignant diseases.

Previous studies have suggested that molecular species larger than the mature calcitonin (CT) are produced by tumors of different origin. In order to study these species, we developed a monoclonal immunoradiometric assay for calcitonin precursors (CT-pr). This assay was based on both monoclonal antibody KC01 directed to the 1-11 region of katacalcin and monoclonal antibody CT08 directed to the 11-17 portion of CT. The sensitivity of this monoclonal immunoradiometric assay for CT-pr was less than 100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels greater than 100 pg/ml; this value was therefore selected as the standard serum value in healthy individuals. CT-pr was present in the serum of seven of ten patients with advanced renal failure and in that of 21 of 52 patients (40%) with benign liver disease but was undetectable in sera of patients with other benign diseases. The serum CT-pr level was correlated with that of mature CT in patients with medullary carcinoma of the thyroid. In contrast, the serum CT-pr level was frequently elevated in the absence of a detectable CT level in patients with various malignant tumors and, particularly, in those with either tumors of the neuroendocrine system (60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumor extract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNA encoding for common region, calcitonin, and katacalcin sequences. These results show that CT precursors are excreted by numerous cancers and might well be useful biological markers for the follow-up of productive tumors.

Drug-induced antibodies during 2-N-methyl-9-hydroxyellipticinium acetate (NSC-264137) treatment: schedule dependency and relationship to hemolysis.

Drug-dependent antibodies were investigated in patients treated with elliptinium acetate, a cytostatic drug with activity in advanced breast cancer. Retrospective analysis of 83 patients, receiving weekly intravenous elliptinium, showed a high incidence of anti-elliptinium antibodies (20%). Hemolysis occurred among antibody-positive patients, apparently related to the antibody titer. The predictability of anti-elliptinium antibodies for hemolysis and the schedule dependency of antibody development was examined prospectively. Among 42 patients treated weekly for at least three courses, 40% developed antibodies. Of 30 patients receiving elliptinium daily for three days every three weeks, none developed either antibodies or hemolysis. Only antibody positive patients, with titers greater than or equal to 32 were at risk for hemolysis. The possible mechanisms are discussed.

Monoclonal antibodies to lipophilic and short-sized haptens: application to the 4-amino-quinoline antimalarial drugs.

Monoclonal antibodies recognizing the 4-amino-7-chloro-quinoline (ACQ) structure, which represents the backbone of the 4-amino-quinoline antimalarial drugs, were obtained in mice, after injection of ACQ coupled to hemocyanin via the glutaraldehyde method. The resulting antibodies show a definite specificity to this hapten, but react better with compounds substituted on the exocyclic amino group in 4. It is postulated that the quinoline ring is not sufficient for the reaction with the antibodies, and that an enlarged structure, which is given by the bridge used to link hapten and carrier, entails an important increase (1000-fold) in the apparent affinity. The striking similarities between this bridge and the lateral chains of the antimalarial drugs are accountable for this enhanced recognition. This result allows us to indicate that in some instances, the bridge-structure of the immunogen should be positively involved in the epitope. This observation may become useful in the conception of immunogens, aiming to obtain antibodies directed against some lipophilic and small-sized haptens.

The immune response to a synthetic peptide analogous to the 109-145 beta hCG carboxyl-terminus is directed against two major and two minor regions.

The immune response to a 37-amino acid synthetic peptide analogous to the carboxyl-terminal part (109-145) of the human chorionic gonadotropin beta subunit (beta hCG) was studied with monoclonal antibodies selected from 31 cell fusion experiments. Analysis of the immunogenic determinants borne on the synthetic peptide (CTP) showed a prevailing response to two immunodominant regions. The first was located on the 110-116 amino acid sequence of the CTP which is also the most hydrophilic region: 50% of anti-CTP antibodies selected for their high binding to 125I beta hCG were directed to this sequence. A second immunodominant portion was recognized by four antibodies, and comprised amino acids 134 to 139, representing a highly O-glycosylated region on the native protein. Moreover, a unique antibody designated FB13 bound to a region located on the last seven amino acids (139-145) of beta hCG. Finally, a hypothetical conformational determinant was recognized by antibody FB02 within the 121-145 region. Thus, the immune response to CTP was directed against two major and two minor regions. These antigenic determinants were demonstrated to be accessible for antibody binding on both the hCG molecule and its beta subunit. Localization of these epitopes suggests a relationship between the hydrophilicity and the immunological potency of different CTP regions.

Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches.

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.

Monoclonal antibodies directed to human and equine chorionic gonadotropins as probes for the topographic analysis of epitopes on the human alpha-subunit.

To improve our knowledge of the structural features of the alpha-subunit of hCG we have studied the antigenic site recognized by monoclonal antibody (MAb) ECG01 raised against equine CG (eCG) which binds to hormones and alpha-subunits from human and equine species. We have also delineated regions of hCG alpha comprising the epitope recognized by HT13 which was raised against hCG and binds to hCG and hCG alpha. To define the residues involved in the antigenic sites recognized by ECG01 and HT13, we have studied the reactivities of these two MAbs with native or chemically modified LH and CG with subunits from equine, human, or ovine (o) species or with synthetic peptides analogous to various portions of hCG alpha. We have also compared these reactivities with those displayed by MAbs AHT20 and FA 36, whose epitopes have been previously described; anti-hCG alpha MAb AHT20 is specific for the free alpha-subunits of various species and recognizes residues localized to the 36-41 region of hCG alpha, whereas antipeptide MAb FA36 binds to the 87-92 carboxyl-terminal part of hCG alpha. Our results show that the epitopes of HT13 and ECG01 are 1) probably discontinuous, as these MAbs did not bind to the reduced and S-carboxymethylated hCG alpha; and 2) constituted by residues borne on the 1-35 and 52-86 sequences, as they do recognize the hCG alpha core missing the 36-51 portion, yet do not recognize hCG alpha-(87-92) region recognized by FA36. The comparative studies performed with specific two-site immunoradiometric assays to determine the interspecies cross-reactivities of the MAbs allow us hypothetical assignment of residues on the primary structure of hCG alpha. The antigenic site recognized by ECG01 might include two to six amino acids, four of these residues being located at inverted places compared to those of oLH alpha (Asp6/Gly22 and Arg67/Lys75). These residues present important charged functional groups highly conserved among evolutionarily related variants, and it is likely that they are located on the surface of both the intact hormone and its alpha-subunit. Three peptidic portions of hCG alpha, 16-17, 64-66, and 73-76, respectively, might be involved in the epitope recognized by HT13, although we could not rule out the possibility that other residues were also involved in the antigenic site. These observations allow us to identify several residues as potentially constituting the epitopes recognized by two MAbs on both hCG and hCG alpha.

Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.

A role for neuropeptide-Y, dynorphin, and noradrenaline in the central control of food intake after food deprivation.

A marked increase in food intake is observed in the rat after central injection of neuropeptide-Y (NPY), dynorphin, or noradrenaline (NA). Levels of both NPY and dynorphin are increased in the hypothalamus of food-deprived rats. The aim of this study was to explore the role of NPY, dynorphin, and NA in the central control of feeding after a period of food deprivation. We have investigated the effect of intracerebroventricular injection of a monoclonal antibody to NPY (NPYAb), a potent and selective kappa-opioid receptor antagonist norbinaltorphimine (norBNI), and the alpha-adrenergic antagonist phentolamine on fast-induced food intake. In animals provided with food after a 24-h fast, NPYAb given 10 min before presentation of food reduced food intake by 30% (P < 0.01) compared to that of animals pretreated with an antibody to chloroquine. A similar (34%; P < 0.05) reduction in fast-induced feeding occurred after pretreatment with norBNI. If norBNI was given together with NPYAb, then a reduction of 51% (P < 0.05) was observed. Pretreatment with phentolamine reduced fast-induced food intake by 39% (P < 0.05), with no evidence of an additive effect when phentolamine was given together with NPYAb. These data would support a role for endogenous NPY, dynorphin, and NA in the mediation of fast-induced feeding. NPY would seem to act independently of dynorphin, but through the same mechanism as NA.

Plasma neuropeptide Y concentrations in patients with neuroendocrine tumors.

In order to develop an immunoradiometric assay for human neuropeptide (hNPY), a recently discovered and potent vasoconstrictor 36-amino-acid peptide, we used hNPY and some of its subpeptides to prepare monoclonal anti-NPY antibodies. Two monoclonal antibodies with high affinity for hNPY, that is affinity constants in the range of 10(10) mol/L-1, which respectively, reacted with the 9-18 portion and the 32-36 portion of hNPY were used in the immunoradiometric system. The assay was highly specific, NPY-related peptides such as pancreatic polypeptide and peptide YY not being detected. The lower limit of sensitivity was 0.5 pmol/L. In 303 normal subjects, plasma NPY concentrations were less than 0.5 pmol/L in 67%, 0.5 to 5.0 pmol/L in 25% and 5.1 to 30 pmol/L in the remaining 8%. A value of 7.5 pmol/L (95th percentile value in the normal group) was considered as the upper limit of normal. Among 111 patients with various neuroendocrine tumors, elevated plasma NPY concentrations were found in patients with pheochromocytomas and neuroblastomas, the highest plasma levels being found in patients with malignant pheochromocytomas. We conclude that patients with neuroendocrine tumors, especially secreting and or malignant tumors of the sympathochromaffin system, often have elevated plasma NPY concentrations.

Sensitive and specific assay for human chorionic gonadotropin (hCG) based on anti-peptide and anti-hCG monoclonal antibodies: construction and clinical implications.

We developed a highly sensitive and specific assay for hCG using monoclonal antibodies (Mabs) directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl-terminus (CTP) of beta hCG. Five antibodies that varied by either their affinity for beta hCG or their specificity for epitopes on CTP were investigated. To measure hormone levels, we used as the radiolabeled indicator an alpha-subunit-reactive Mab. The monoclonal-immunoradiometric assay had a lower limit of sensitivity of 0.05 ng/ml. Serum levels of hCG or hCG-like material with CTP structure were measured in 229 healthy blood donors; 1.1% of healthy men and 4.6% of nonpregnant women younger than 50 yr had serum values varying between 0.05 and 0.23 ng/ml. Moreover, 6 to 7 healthy women older than 50 yr had detectable levels in the 0.05-0.20 ng/ml range. To study the disappearance rates in normal women, we followed serum hCG serum levels of 6 women who had previously received a single im injection of the hormone. These individuals failed to develop a pregnancy after in vitro fertilization; hCG declined from 0.5 to 0.05 ng/ml within 2 weeks. These results were in contrast to the findings in 12 patients with hCG-producing tumors. In 9 patients without any evidence of recurrent disease, hCG levels became undetectable within 5 months. However, 3 others had levels consistently above 0.05 but below 0.5 ng/ml. In 2 of these three patients, subsequent increasing hCG levels were associated with tumor recurrence. We conclude that this hCG assay based on both anti-peptide and anti-hCG Mabs may be useful in tumor monitoring.

Procalcitonin increase after endotoxin injection in normal subjects.

As procalcitonin concentrations have been shown to be elevated in patients with septicemia and gram-negative infections in particular, we proceeded to investigate the effect of endotoxin, a product of gram-negative bacteria, on procalcitonin concentrations in normal human volunteers. Endotoxin from Escherichia coli 0113:H10:k, was injected i.v. at a dose of 4 mg/kg BW into these healthy volunteers. Blood samples were obtained before and 1, 2, 4, 6, 8, and 24 h after injection of the endotoxin. Each patient's cardiovascular and overall clinical status was monitored over this period. The patients developed chills and rigors, myalgia, and fever between 1-3 h. Tumor necrosis factor-alpha levels increased sharply at 1 h and peaked at 90 min, reaching the baseline concentration thereafter by 6 h. Interleukin-6 levels increased more gradually, peaking at 3 h and reaching the baseline concentration at 8 h. The procalcitonin concentration, which was undetectable (< 10 pg/mL) at 0, 1, and 2 h, was detectable at 4 h and peaked at 6 h, maintaining a plateau through 8 and 24 h (4 ng/mL). There was no elevation of calcitonin concentrations, which remained below 10 pg/mL, the lowest sensitivity of the assay. Procalcitonin was measured by a two-antibody immunoradiometric assay specific for this peptide, with no cross-reactivity with calcitonin, katacalcin, or calcitonin gene-related peptide. We conclude that endotoxin induces the release of procalcitonin systemically, that this increase is not associated with an increase in calcitonin, and that the increase in procalcitonin associated with septicemia in patients may be mediated through the effect of endotoxin described here. Whether procalcitonin participates in the mechanisms underlying inflammation remains to be investigated.

Production and in vitro utilization of monoclonal antibodies to human thyroglobulin.

Human thyroglobulin (Tg) was used as an antigen in the development of antibodies by the hybridoma technique. From four antibodies that bound more than 40% labeled Tg, two were characterized (182/E4 and 211/A5). They were both of the immunoglobulin G 2ab subclass, and provided an affinity constants (Ka) of 1.2 X 10(10) and 7.7 X 10(9) mol-1, respectively. The specificity of these antibodies was demonstrated by the absence of cross-reaction by monoiodothyronine, diiodothyronine, T3, T4, and sialic acid. A RIA was developed with 182/E4 or 211/A5, and the least detectable dose, based on the standard curve, was 10 ng/ml. The immunoreactivities of 182/E4 and 211/A5 to four Tg preparations different in iodine content appeared to be identical. Histochemical staining was used on normal and neoplastic tissues with both antibodies. Positive reactions were obtained in both cells and colloid, with heterogeneous staining from one follicle to another. Papillary carcinoma showed numerous positive cells, in contrast with Hürtle cell tumors which displayed very few positive cells. Anaplasic giant and spindle cells were negative. Monoclonal antibodies to human Tg are useful for in vitro detection of Tg.

Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV).

Increased concentrations of procalcitonin (PCT) are found in the plasma of patients with thermal injury and in patients with sepsis and severe infection, making this molecule important as a diagnostic and prognostic marker in these diseases. Interestingly, only the truncated form of PCT, PCT(3-116), is present in the plasma of these patients. The enzyme responsible for this truncation is unknown as yet. Here, using capillary zone electrophoresis, mass spectrometry and Edman sequence analysis, we demonstrate that dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) is capable of catalyzing the hydrolysis of PCT(1-116), releasing the N-terminal dipeptide Ala-Pro. We hypothesize that PCT(3-116) is the result of the hydrolysis of PCT(1-116) by soluble DP IV of the blood plasma or by DP IV expressed on the surface of cells.

Purification and properties of human erythrocyte delta-amino-levulinic acid dehydratase (EC 4-2-1-24).

Human delta-aminolevulinic acid dehydratase (ALA-D) was purified 9 000-fold by salt precipitation, ion-exchange chromatography and gel filtration. These methods resulted into an electrophoretically and immunologically pure protein. The optimum pH of the enzyme is 6.6 and its Km with ALA : 4.8 X 10(-4) M. The enzymatic activity was increased by thiol-containing substances, such as dithiothreitol (DTT), which protect the -SH groups of the protein. Zinc, a portion of the enzyme molecule, was partly lost during the purification procedure; its addition enhances the enzymatic activity. Determination of molecular weights and electron microscopy study are in favor of an octameric structure.

Antibody recognition of substituted ammonium ions. Modulation by the counterion.

Antibodies directed against elliptinium acetate, a quaternary ammonium compound with antineoplastic activity in man, were obtained in rabbits, after conjugation of the drug with haemocyanin. These antibodies are specific for the quaternary ammonium structure. However, the recognition of the drug can be markedly decreased (ten-fold) by changing the associated counterion. These observations were extended to other ellipticine derivatives that exist in two forms: acetate and chloride. In each case, the recognition of the acetate form was 7-11-fold higher than that of the chloride form. These results could be explained by high-energy strengths existing between the cation and the anion, resulting in a paired-ion antigen. This represents the first identification of antibodies directed to a paired-ion structure, with specificity for both the cation and anion used for immunization. Such results are relevant in the construction of immunoassays for quaternary ammonium compounds.

Characterization of two monoclonal antibodies against the COOH-terminal part of the human epidermal growth factor receptor and potential clinical use.

Two monoclonal antibodies of the IgG2 subclass, designated A 01 and B 11, were prepared against two synthetic peptides corresponding to the COOH-terminal sequence of the human epidermal growth factor receptor (EGF-R), in order to detect EGF-R in a radioimmunometric assay and by immunohistochemistry. Characterization of these Mabs showed that they recognized two different eptiopes on the original peptides with Kd of 1.7 x 10(-8) M and 1.3 x 10(-7) M, respectively, without crossreaction. The A 431 antigen recognized by A 01 and B 11 had an apparent molecular weight of approximately 170,000 and was able to specifically link to EGF. Thus, A 01 and B 11 are directed against an antigenic site on the human EGF-R. With Western blot analysis and immunostaining, A 01 was shown to be EGF-R specific. In addition to the EGF-R, B 11 recognized two unidentified soluble proteins present in the cytoplasm of the SKBR-3 cell line but different from the c-erb B-2 oncoprotein expressed by these cells. Mabs A 01 and B 11 were used in an IRMA for the determination of EGF-R using the A 431 cell line as a source of EGF-R. Mab A 01 was also shown to be a useful tool for immunohistochemical detection of EGF-R.