Pinhole micro-SPECT/CT for noninvasive monitoring and quantitation of oncolytic virus dispersion and percent infection in solid tumors.

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). Sodium iodide symporter (NIS) RNA level and dispersion pattern were determined in control and MV-NIS-infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with (123)I or (99)TcO(4) micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r(2)=0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS-infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity.

We have defined a new paraneoplastic immunoglobulin G (IgG) autoantibody specific for CRMP-5, a previously unknown 62-kd neuronal cytoplasmic protein of the collapsin response-mediator family. CRMP-5 is in adult central and peripheral neurons, including synapses, and in small-cell lung carcinomas. Since 1993, our Clinical Neuroimmunology Laboratory has detected CRMP-5-IgG in 121 patients among approximately 68,000 whose sera were submitted for standardized immunofluorescence screening because a subacute neurological presentation was suspected to be paraneoplastic. This makes CRMP-5 autoantibody as frequent as PCA-1 (anti-Yo) autoantibody, second only to ANNA-1 (anti-Hu). Clinical information, obtained for 116 patients, revealed multifocal neurological signs. Most remarkable were the high frequencies of chorea (11%) and cranial neuropathy (17%, including 10% loss of olfaction/taste, 7% optic neuropathy). Other common signs were peripheral neuropathy (47%), autonomic neuropathy (31%), cerebellar ataxia (26%), subacute dementia (25%), and neuromuscular junction disorders (12%). Spinal fluid was inflammatory in 86%, and CRMP-5-IgG in 37% equaled or significantly exceeded serum titers. Lung carcinoma (mostly limited small-cell) was found in 77% of patients; thymoma was in 6%. Half of those remaining had miscellaneous neoplasms; all but two were smokers. Serum IgG in all cases bound to recombinant CRMP-5 (predominantly N-terminal epitopes), but not to human CRMP-2 or CRMP-3.

Nicotinic acetylcholine receptors of muscle and neuronal (alpha7) types coexpressed in a small cell lung carcinoma.

SCC-37 is a small cell lung carcinoma line that aberrantly expresses muscle-type nicotinic acetylcholine receptors (nAChRs). It was established from a patient with a paraneoplastic autoimmune neuromuscular disorder, myasthenia gravis. When grown as a xenograft tumor, SCC-37 cells express plasma membrane receptors that bind 1251-labeled alpha-bungarotoxin (125I-alpha-BTx), cosediment with 9S nAChR pentamers, and bind to a monoclonal antibody (MAb 35) specific for muscle-type (alpha1 subunit) alpha-BTx receptors. The agonist carbamylcholine (carbachol) stimulates influx of 22Na+ in SCC-37 cells; this is inhibited by alpha-BTx and by d-tubocurarine. Long-term cultured SCC-37 cells have functional and ligand-binding evidence for surface coexpression of both alpha1 and neuronal-type (alpha7 subunit) alpha-BTx receptors. A subclone of SCC-37, designated SCC-A9, expresses only the neuronal-type (alpha7 subunit) alpha-BTx receptors on its surface. Carbachol does not stimulate 22Na+ influx in SCC-A9 cells, but cytisine initiates a sustained influx of Ca2+. Activation of this response is inhibited by alpha-BTx and by the alpha7-selective antagonist methyllycaconitine. Addition of Co2+ abrogates the sustained elevation of intracellular free Ca2+ concentration, implying that the cytisine-stimulated influx of Ca2+ is sustained by secondary opening of voltage-sensitive channels in the plasma membrane. Surface receptors for 125I-alpha-BTx are blocked by methyllycaconitine and d-tubocurarine. Solubilized alpha-BTx receptors from plasma membranes of SCC-A9 cells cosediment with 10S neuronal nAChR pentamers and bind to an alpha7-specific monoclonal antibody (MAb P27) but not to the muscle nAChR-reactive MAb 35. However, MAb P27 and MAb 35 both bind to alpha-BTx receptors solubilized from the cytoplasmic compartments of SCC-A9 and the parental SCC-37 line. Reverse transcription-PCR analysis revealed RNA transcripts for alpha7 and alpha1 subunits in both SCC-A9 and SCC-37 cells. The nAChRs that are expressed in these novel human cell lines can regulate cation fluxes directly as well as indirectly by synergizing with the activity of voltage-sensitive Ca2+ channels. These activities may influence the secretion of autocrine growth factors and the transcription of growth regulatory genes and thus be pertinent to the growth and metastasis of malignant neuroendocrine neoplasms.

Calcium-channel antibodies in the Lambert-Eaton syndrome and other paraneoplastic syndromes.

BACKGROUND: Voltage-gated calcium channels in small-cell lung carcinomas may initiate autoimmunity in the paraneoplastic neuromuscular disorder Lambert-Eaton syndrome. The calcium-channel subtype that is responsible is not known. METHODS: We compared the effects of antagonists of L-type, N-type, and P/Q-type neuronal calcium channels on the depolarization-dependent influx of calcium-45 in cultured carcinoma cells. Serum samples from patients with various disorders were tested for reactivity with P/Q-type channels solubilized from carcinoma and cerebellar membranes and N-type channels from cerebral cortex. RESULTS: P/Q-type calcium-channel antagonists were the most potent inhibitors of depolarization-induced 45Ca influx in cultured small-cell carcinoma cell lines. Anti-P/Q-type calcium-channel antibodies were found in serum from all 32 patients with Lambert-Eaton syndrome and a diagnosis of cancer and in 91 percent of the 33 patients with Lambert-Eaton syndrome without cancer. Anti-N-type calcium-channel antibodies were found in 49 percent of the 65 patients with the Lambert-Eaton Syndrome. Lower titers of anti-P/Q-type and anti-N-type calcium-channel antibodies were found in 54 percent of 70 patients with a paraneoplastic encephalomyeloneuropathic complication of lung, ovarian, or breast carcinoma, 24 percent of 90 patients with cancer but no evident neurologic complications, 23 percent of 78 patients with sporadic amyotrophic lateral sclerosis, and less than 3 percent of 69 patients with myasthenia gravis, epilepsy, or scleroderma. CONCLUSIONS: The high frequency of P/Q-type calcium-channel antibodies found in patients with Lambert-Eaton syndrome implies that antibodies of this specificity have a role in the presynaptic pathophysiology of this disorder.

Nine residues influence the binding of alpha-bungarotoxin in alpha-subunit region 185-200 of human muscle acetylcholine receptor.

Identification of residues in the skeletal muscle nicotinic acetylcholine receptor (AChR) that bind snake venom alpha-neurotoxin antagonists of acetylcholine [e.g., alpha-bungarotoxin (alpha-BTx)] provides structural information about the neurotransmitter binding region of the receptor. Using synthetic peptides of the human AChR alpha-subunit region 177-208, we previously localized a pharmacologically specific binding site for alpha-BTx in segment 185-199. To define in more detail the residues that influence the binding of alpha-BTx to this region, we prepared 16 peptide analogues of the alpha-subunit segment 185-200, with the amino acid L-alanine sequentially replacing each native amino acid. Circular dichroism spectroscopy did not reveal changes in the secondary structure of the peptides except for the analogue in which Pro194 was substituted with alanine. This implies that any change in alpha-BTx binding could be attributed to replacement of the native residue's side chain by alanine's methyl group, rather than to a change in the structure of the peptide. The influence of each substitution with alanine was determined by comparing the analogue to the parental sequence alpha 185-200 in solution-phase competition with native human AChR for binding of 125I-labeled alpha-BTx. The binding of alpha-BTx by analogue peptides with alanine substituted for Tyr190, Cys192, or Cys193 was greatly diminished. Binding of alpha-BTx to peptides containing alanine replacements at Val188, Thr189, Pro194, Asp195, or Tyr198 was also reduced significantly (p < 0.003). An unanticipated finding was that substitution of alanine for Ser191 significantly increased alpha-BTx binding (p < 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular diversity of neuronal-type calcium channels identified in small cell lung carcinoma.

Using the polymerase chain reaction (PCR), we identified RNA transcripts for two distinct classes of neuronal-type voltage-gated Ca2+ channels (VGCC) in a prototypic small cell lung carcinoma (SCLC) cell line, SCC-9. Oligonucleotide primers were designed to encode amino acid sequences common to alpha 1-subunits of known neuronal VGCC classes. Sequencing of complementary DNA (cDNA) clones derived from two independent PCR products revealed that one corresponded to a brain class A VGCC fragment predicted to encode a P-type VGCC (insensitive to dihydropyridines and omega-conotoxin) characteristic of cerebellar Purkinje cells but not previously identified in humans. The second PCR product was identical (except for one conservative nucleotide difference) to a fragment of the class D VGCC of neurons and neuroendocrine cells, which encodes an L-type VGCC (sensitive to dihydropyridines). By Northern blot analyses, both cDNAs hybridized to messenger RNAs (mRNAs) obtained from SCC-9; class D hybridized additionally to human cerebral cortical mRNA, but neither hybridized to mRNA from the skeletal muscle cell line TE671. Although no cDNA corresponding to class B VGCC (N-type) was identified, SCLCs are known to express VGCC that are sensitive to omega-conotoxin and coprecipitate with 125I-labeled-omega-conotoxin when complexed with serum IgG from patients with the Lambert-Eaton myasthenic syndrome. The multiple classes of neuronal-type VGCC expressed in SCLC could conceivably have both unique and related antigenic determinants that may give rise to antineuronal autoimmune responses. This would account for a spectrum of paraneoplastic neurologic disorders including the Lambert-Eaton syndrome and subacute cerebellar degeneration.

Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor alpha subunit extracellular domain 1-210.

The duplicated alpha subunits account for 40% of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rH alpha 1-210) corresponding to the proposed extracellular domain of the alpha subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human alpha subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rH alpha 1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57-72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's alpha subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells.

An avidin-biotin-peroxidase assay to detect synthetic peptides bound to polystyrene plates.

The simple chemical method described for detecting synthetic peptides bound to polystyrene should facilitate interpretation of studies involving interactions of antibodies or solubilized major histocompatibility complex (MHC) molecules with immobilized peptide antigens. After its application to an ELISA plate, the peptide is biotinylated in situ and avidin-biotinylated horseradish peroxidase complex and substrate are added sequentially. For 36 of 43 peptides tested (11-27 residues long), a colored reaction product confirmed that the peptide was bound. In three of the seven instances of a negative result, peptides were positively detected by binding of antibody. Four instances remained in which it could not be determined whether the peptide did not bind to the plate or whether it was not biotinylated. On the other hand, the biotin-ABC assay positively detected peptide in 18 of 22 instances without evidence of antibody binding, implying seronegativity or a loss of antigenic conformation in the bound peptide. This general method should be applicable to assays of microbial antigens, autoantigens and allergens. Modification of the technique by use of biotin hydrazide should enable monitoring of the binding to polystyrene of carbohydrates, glycolipids or DNA.

Alpha-bungarotoxin binds to human acetylcholine receptor alpha-subunit peptide 185-199 in solution and solid phase but not to peptides 125-147 and 389-409.

The nicotinic acetylcholine receptor (AChR) of human skeletal muscle has a reducible disulfide bond near the neurotransmitter binding site in each of its alpha-subunits. By testing a panel of overlapping synthetic peptides encompassing the alpha-subunit segment 177-208 (containing cysteines 192 and 193) we found that specific binding of 125I-labelled alpha-bungarotoxin (alpha-BTx) was maximal in the region 185-199. Binding was inhibited by unlabelled alpha-BTx greater than d-tubocurarine greater than atropine greater than carbamylcholine. Peptide 193-208 did not bind alpha-BTx, whereas 177-192 retained 40% binding activity. Peptides corresponding to regions 125-147 (containing cysteines 128 and 142) and 389-409, or peptides unrelated to sequences of the AChR failed to bind alpha-BTx. No peptide bound 125I-alpha-labelled parathyroid hormone. The apparent affinity (KD) of alpha-BTx binding to immobilized peptides 181-199 and 185-199 was approximately 25 microM and 80 microM, respectively, in comparison with alpha-BTx binding to native Torpedo ACh receptor (apparent KD approximately 0.5 nM). In solution phase, both peptides effectively competed with solubilized native human AChR for binding of alpha-BTx, and peptide 185-199 showed little evidence of dissociation after 24 h. Peptides that bound alpha-BTx did so when sulfhydryls were reduced. Cysteine modification, by N-ethylmaleimide or acetamidomethylation, abolished alpha-BTx-binding activity. The data implicate the region of cysteines 192 and 193 in the binding of neurotransmitter to the human receptor.

Evidence against acetylcholine receptor having a main immunogenic region as target for autoantibodies in myasthenia gravis.

We investigated specificities of acetylcholine receptor (AChR) antibodies in 100 seropositive patients with myasthenia gravis (MG). Antibodies in 74 of these sera were inhibited by more than 50% from binding to human muscle AChR by a rat monoclonal antibody (mAb) of "main immunogenic region" (MIR) specificity. The mAb inhibition was not explainable by epitope competition because (1) the mAb was reactive with both Torpedo and human AChR, but antibodies in 85 of the MG sera did not bind to Torpedo AChR, and (2) the mAb blocked binding of rat anti-peptide antibodies to an alpha subunit region of the human AChR unrelated antigenically to the designated MIR region. Individual patients' sera had evidence of extensive antibody heterogeneity and revealed interspecies polymorphisms in AChR antigenicity, near and remote from the neurotransmitter-binding region. The data challenge the concept that a MIR of the AChR is the principal stimulus for antibody production in MG and emphasize a potential pitfall in assuming seronegativity in MG on the basis of a single assay system.

Antagonism of voltage-gated calcium channels in small cell carcinomas of patients with and without Lambert-Eaton myasthenic syndrome by autoantibodies omega-conotoxin and adenosine.

The Lambert-Eaton myasthenic syndrome (LES) is an autoimmune presynaptic disorder of peripheral cholinergic neurotransmission in which there is often an associated small cell lung carcinoma (SCC). SCC lines established from patients with and without LES exhibit a Ca2+ influx response to depolarization by K+ that is consistent with the presence of voltage-gated Ca2+ channels. Autoantibodies antagonistic to SCC Ca2+ channel activity were found exclusively in patients with LES, independent of cancer status. Depolarization-induced uptake of 45Ca2+ by SCC lines was reduced maximally after 3-4 days of exposure to serum immunoglobulins from 14 of 19 LES patients, while 53 control immunoglobulins (including patients with SCC, other tumors, other paraneoplastic syndromes, and other neurological and autoimmune diseases) were without effect. The snail neurotoxin omega-conotoxin of subtype GVIA, which is a specific antagonist of presynaptic Ca2+ channels, inhibited K+-stimulated Ca2+ uptake in a dose-dependent manner that was essentially irreversible. Adenosine, reported to be a specific antagonist of neuronal Ca2+ channels, also impaired voltage-stimulated Ca2+ influx in SCC. Use of LES patients' IgG and omega-conotoxin in further studies of SCC may facilitate identification and purification of the LES antigen(s) and yield a quantitative serological test for diagnosing this autoimmune paraneoplastic syndrome.

Myasthenogenicity of human acetylcholine receptor synthetic alpha-subunit peptide 125-147 does not require intramolecular disulfide cyclization.

This study reports the synthesis of a disulfide-looped peptide corresponding to residues 125-147 (Cys 128-Cys 142) of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle, H alpha 125-147 (Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln- Asn-Cys-Ser-Nle-Lys Leu-Gly), and a nondisulfide-looped analogue, H alpha 125-147(S) (Lys-Ser-Tyr-Ser-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu- Gln-Asn-Cys-Ser-Nle-Lys-Leu-Gly), in which the amino acid Cys 128 was replaced with serine. Both peptides induced antigen-specific helper T cell responses, as evidenced in vitro by lymph node cell proliferation and in vivo by production of anti-AChR antibodies. Rats immunized with 100 micrograms of either synthetic peptide, without conjugation to a carrier, produced anti-peptide antibodies which bound to native AChR in immunoprecipitation assays and induced modulation of membrane-bound AChR from cultured human myotubes. Both peptides also induced electrophysiologic and biochemical signs of experimental autoimmune myasthenia gravis. Thus, region 125-147 of the AChR alpha-subunit is at least partly exposed extracellularly in human muscle and contains one or more autoantigenic sites capable of stimulating T cells and B cells. Disulfide-linkage between residues Cys 128 and Cys 142 is not essential for myasthenogenicity.

Region of peptide 125-147 of acetylcholine receptor alpha subunit is exposed at neuromuscular junction and induces experimental autoimmune myasthenia gravis, T-cell immunity, and modulating autoantibodies.

A major antigenic region of native nicotinic acetylcholine receptors (AcChoR) has been identified by using a synthetic disulfide-looped peptide corresponding to alpha-subunit residues 125-147 of Torpedo electric organ AcChoR: Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe- Pro-Phe-Asp-Gln-Gln-Asn-Cys-Thr-Met-Lys-Leu-Gly. The peptide bound 26-56% of polyclonal antibodies induced in rat, rabbit, and dog by immunization with native AcChoR. Rats inoculated with 50 micrograms of unconjugated peptide developed helper T-cell responses, delayed hypersensitivity, and antibodies to native AcChoR. Anti-peptide antibodies were more reactive with native than denatured AcChoR and bound to the alpha subunit. Some reacted exclusively with mammalian muscle AcChoR, some induced modulation of AcChoR on cultured myotubes, but none inhibited binding of alpha-bungarotoxin to solubilized or membrane-associated AcChoR. Repeated immunization induced experimental autoimmune myasthenia gravis: clinical signs in one rat and electrophysiologic and/or biochemical signs in 10 of 11 rats. Thus, at least part of the corresponding region of the mammalian AcChoR alpha subunit is extracellular at the neuromuscular junction and a potential target for pathogenic autoantibodies in patients with acquired myasthenia gravis.

Zinc metabolism in testicular feminization and surgical cryptorchid testes in rats.

Rats with testicular feminization (Tfm) had been reported to have a testis specific zinc deficiency. In this report it is demonstrated that this organ specific zinc deficiency was not corrected by intraperitoneal zinc administration. Normal littermates on the other hand showed a positive testicular response to zinc administration. The increased testicular zinc level in control animals returned to normal 1 week after the zinc treatment probably due to the fast turnover of this element in the testis. Not only surgically induced cryptorchidism but also surgical cryptorchidism and epididymodeferentectomy (to simulate Tfm conditions in normal adult rats) caused a drastic reduction in testicular zinc level. Unlike in Tfm rats, however, the decrease in zinc content in operated animals was not accompanied by a corresponding decrease in alkaline phosphatase activity. Zinc concentration and alkaline phosphatase activity in plasma or other organs were not affected by the surgical procedure. The testicular copper content in the operated animals was higher than that of the unoperated controls.

Copper and ceruloplasmin activity in human amniotic fluid.

The variation in the level of copper and ceruloplasmin oxidase activity in human amniotic fluid from 20 weeks' gestation to term was reported. The protein content of amniotic fluid decreased toward term. A definite decreasing trend of concentration of copper, expressed both as nanograms of copper per milliliter of amniotic fluid and nanograms of copper per milligram of protein, was also observed from midgestation toward term. Ceruloplasmin, on the other hand, demonstrated a significant increase from the period 20 38 weeks' gestation, with a subsequent decline after 38 weeks.