Circulating Agonist Autoantibody to 5-Hydroxytryptamine 2A Receptor in Lean and Diabetic Fatty Zucker Rat Strains.

AIMS: Circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor were increased in older adult type 2 diabetes in association with certain neurodegenerative complications. The male Zucker diabetic fatty (ZDF) rat is a model system for studies of obese, type 2 diabetes mellitus. The aim of the current study was to test for (and compare) circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor in the Zucker diabetic fatty rat and age-matched lean Zucker rat strains. METHODS: Plasma from lean and Zucker diabetic fatty rat (obtained at different developmental stages) was subjected to protein G affinity chromatography. The resulting immunoglobulin G fraction was tested for neurotoxicity (acute neurite retraction, accelerated neuron loss) in N2A mouse neuroblastoma cells and for binding to a linear synthetic peptide corresponding to the second extracellular loop of the 5-hydroxytryptamine 2A receptor. RESULTS: The male Zucker diabetic fatty rat (fa/fa) and two Zucker lean strains (+/?) and (fa/+) harbored autoantibodies to the 5-hydroxytryptamine 2A receptor which appeared spontaneously around 7-8.5 weeks of age. The circulating autoantibodies persisted until at least 25 weeks of age in the Zucker diabetic fatty rat and in the Zucker heterozygote (fa/+), but were no longer detectable in 25-week-old lean (+/?) Zucker rats. Autoantibody-induced acute neurite retraction and accelerated loss in mouse neuroblastoma N2A cells was dose-dependently prevented by selective antagonists of the 5-hydroxytryptamine 2A receptor. It was also substantially prevented by co-incubation with antagonists of RhoA/Rho kinase-mediated signaling (Y27632) or Gq11/phospholipase C/inositol triphosphate receptor-coupled signaling. CONCLUSIONS: These data suggest that neurotoxic 5-hydroxytryptamine 2A receptor-targeting autoantibodies increase in the aging male Zucker diabetic fatty rat and in male Zucker lean rats harboring a heterozygous mutation, but not in age-matched, older Zucker lean rats lacking a known leptin receptor mutation. The Zucker genetic strain may be useful in studies of the role of humoral and/or innate immunity in late neurodegeneration.

Increased basic fibroblast growth factor-like substance in plasma from a subset of middle-aged or elderly male diabetic patients with microalbuminuria or proteinuria.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell growth factor that does not normally circulate in healthy nonpregnant adults. bFGF has been reported in plasma from patients with certain tumors consistent with a postulated role in tumor angiogenesis. In the present study we used an endothelial cell bioassay to test for a bFGF-like substance in plasma and urine from patients with noninsulin-dependent diabetes mellitus. We found increased bFGF immunoreactivity that correlated with bFGF-like endothelial cell growth-promoting activity in plasma from a subset of diabetic patients with persistent microalbuminuria or overt proteinuria. Plasma (bFGF-like) growth-promoting activity was significantly correlated with glycosylated hemoglobin (P < 0.05), but not patient age, race, degree of proteinuria, or systolic blood pressure. In a group of microalbuminuric or proteinuric diabetic subjects well matched according to baseline clinical characteristics, plasma (bFGF-like) growth-promoting activity was significantly decreased (P < 0.0001) in the subgroup of patients who were being treated with an angiotensin-converting enzyme inhibitor simultaneous to blood drawing for plasma growth assay. In patients not treated with an angiotensin-converting enzyme inhibitor, multiple regression analysis showed that retinopathy was the only variable significantly associated with plasma growth-promoting activity. These results imply that plasma bFGF endothelial cell growth-promoting activity is increased and may contribute to pathophysiology in a heterogeneous subset of noninsulin-dependent diabetes mellitus patients with persistent microalbuminuria or overt proteinuria.

Circulating fibroblast growth factor-like autoantibodies in two patients with multiple endocrine neoplasia type 1 and prolactinoma.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. bFGF lacks a classical amino-terminal signal sequence and is not readily detectable in plasma from normal subjects. In earlier studies we showed increased bFGF-like mitogenic activity for parathyroid-derived endothelial cells and (increased) bFGF immunoreactivity (0.24-1.28 ng/mL) in plasma of subjects with multiple endocrine neoplasia type 1 (MEN-1). In the present study we examined the proliferative activity of MEN-1 and normal plasmas (applied to protein-A columns) in calf pulmonary artery endothelial cells. Protein-A-eluted activity in plasma from MEN-1 prolactinoma plasma exceeded activity from normal and MEN-1 nonprolactinoma plasma in three of eight MEN-1 subjects with untreated or recurrent prolactinoma. Protein-A-eluted active fractions from MEN-1 prolactinoma plasma had several properties of an immunoglobulin G, including affinity for antihuman immunoglobulin G (IgG) agarose, sensitivity to thiols, and (prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions) apparent mol wt corresponding to those of the heavy and light chains of IgG. The IgG fraction of MEN-1 prolactinoma plasma had far more activity in endothelial cells than did optimal concentrations of known growth factors or conditioned medium from prolactinoma cells. Endothelial cell bioactivity in protein-A-eluted fractions from MEN-1 prolactinoma plasma was neutralized 70% by rabbit antibodies to intact bFGF. These results imply novel growth stimulatory bFGF-like autoantibodies in a subset of MEN-1 patients with prolactinoma.

Increased basic fibroblast growth factor in plasma from multiple endocrine neoplasia type 1: relation to pituitary tumor.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. We previously reported a basic fibroblast growth factor (bFGF)-like substance in the plasma of subjects with MEN1. In the present study we used a novel sensitive specific 2-site immunoradiometric assay to test for bFGF in plasma. The assay employs immobilized affinity-purified N-terminal-specific anti-bFGF antibodies (antigen capture) and high affinity binding to radioiodinated heparin. bFGF-like immunoreactivity was undetectable (< 0.2 ng/mL) in normal subjects and in most unaffected relatives of MEN1 subjects. We found detectable bFGF ranging from 0.24-1.28 ng/mL in 21 of 50 subjects with MEN1. Seven of 8 MEN1 subjects with untreated pituitary tumors had detectable plasma bFGF-like immunoreactivity. Plasma bFGF-like immunoreactivity decreased after surgery for pituitary tumor in 4 patients and after initiation of bromocryptine therapy in 4 patients. bFGF was increased in the plasma of several subjects with sporadic endocrine disorders, including 3 with untreated or persistent acromegaly. We conclude that pituitary tumor is a possible source of high circulating bFGF immunoreactivity in MEN1 plasma.

Studies in a kindred with parathyroid carcinoma.

We report a family with primary hyperparathyroidism in four patients in two generations with apparent autosomal dominant transmission. A fifth member was probably affected. Two cases had definite parathyroid carcinoma (PC), and two had parathyroid adenoma with atypical features that could represent an early stage of cancer. In each of our patients, one parathyroid gland was abnormal. Five other parathyroid glands (in two patients) were normal in histology and size. There was no evidence of neoplasia in other tissues. Constitutional karyotypes were normal in all four patients. We identified three chromosomal abnormalities (a reciprocal translocation between chromosomes 3 and 4, trisomy 7, and a pericentric inversion in chromosome 9) in cultured PC tissue from one patient. These chromosomal changes are of unclear significance. Analyses on tumor DNA from one case of PC and one of atypical adenoma showed no evidence of ras gene mutations, PTH gene rearrangement, or allelic loss from chromosome 11q13 (locus of the gene for multiple endocrine neoplasia type 1). This family shows susceptibility to cancer without antecedent hyperplasia in all parathyroids. It could help identify a novel tumor susceptibility gene.

Locally recurrent parathyroid neoplasms as a cause for recurrent and persistent primary hyperparathyroidism.

Between 1982 and 1989, 145 patients underwent operations for persistent or recurrent primary hyperparathyroidism (HPT). At re-exploration, 15 patients (10.3%) were found to have locally recurrent parathyroid tumors (11 patients with adenoma and 4 with carcinoma). These 15 patients had 28 previous operations at outside institutions for HPT. Patients with locally recurrent HPT secondary to adenoma had a longer disease-free interval than patients with locally recurrent carcinoma. At the time of evaluation at the National Institutes of Health (NIH) for recurrent or persistent HPT, each patient was symptomatic and patients with carcinoma had significantly more symptoms and higher serum levels of calcium and parathyroid hormone than patients with adenoma. Locally recurrent parathyroid neoplasm was correctly localized by preoperative testing in 14 of 15 patients. These 15 patients underwent 18 reoperations at NIH for excision of locally recurrent parathyroid tumors. Following the final reoperation (two patients had more than one procedure), each patient had normal serum levels of calcium. In addition each patient remains biochemically cured (based on normal serum calcium level), with a median follow-up interval of 21 months. Local recurrence of parathyroid adenoma comprises a small but significant proportion of cases of recurrent or persistent HPT and can be indistinguishable from parathyroid carcinoma. Findings suggestive of carcinoma include shorter disease-free interval, higher serum levels of calcium and parathyroid hormone, and histologic appearance. Whether the locally recurrent parathyroid neoplasm is benign or malignant, aggressive surgery can control serum levels of calcium in these patients with acceptable rates of morbidity.

Circulating fibroblast growth factor-like substance in familial multiple endocrine neoplasia type 1.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. Basic FGF lacks a classical signal sequence, and it is not clear how it is released from cells. bFGF or bFGF-like activity has not been previously demonstrated in plasma. In an earlier study we showed increased mitogenic activity for parathyroid-derived epithelial and mesenchymal cells in plasma of subjects with familial multiple endocrine neoplasia type 1 (FMEN1). In the present study we examined the growth-promoting activity of normal and FMEN1 plasmas [applied to heparin-Sepharose (HS) columns] in parathyroid-derived cloned endothelial cells. FMEN1 plasma HS-adsorbed activity exceeded normal plasma HS-adsorbed activity in 6 of 8 FMEN1 plasma samples. Peak (FMEN1 plasma) HS-adsorbed activity eluted with 0.1-0.3 M NaCl, was completely neutralized by specific antibodies against bFGF, and had an apparent mol wt of 110 kD. Active fractions from FMEN1 plasma prepared by gel filtration in 7 M urea displayed apparent mol wt of about 14-16 kD and showed increased apparent affinity for HS; recovered activity appeared principally in the 3.0-M NaCl eluate. Using a sensitive two-site immunoradiometric assay for bFGF we found 0.4 ng/mL bFGF-like immunoreactivity in the highly purified 3.0-M NaCl eluate from a HS column to which the active components from gel filtration of FMEN1 plasma in 7 M urea were applied. These results imply that bFGF or closely related factors circulate in FMEN1.

Clonality of parathyroid tumors in familial multiple endocrine neoplasia type 1.

Familial multiple endocrine neoplasia type 1 (MEN-1) is characterized by tumors of the parathyroids, endocrine pancreas, and anterior pituitary. Since the gene associated with MEN-1, located on chromosome 11 (11q13), may normally inhibit tumor proliferation, tumors could arise from inactivation of one or both of the alleles. However, parathyroid tumors in patients with MEN-1 have been considered to result from polyclonal hyperplasia. Using genetic probes, we tested parathyroid tumors for a monoclonal component, represented by a loss of alleles at any of eight loci along chromosome 11. Ten of 16 tumors from 14 patients with familial MEN-1 had losses of alleles from chromosome 11. Tumors with losses were larger than those without (1.6 vs. 0.2 g; P less than 0.002), suggesting that a monoclonal adenoma may develop after a phase of polyclonal hyperplasia. In 7 of 10 tumors, the subregion of loss was less than the full length of chromosome 11 but always included one copy of the MEN-1 locus. Of 34 sporadic adenomas from patients without MEN-1, 9 showed similar allelic losses in chromosome 11; in 7 the losses included the apparent MEN-1 locus. We conclude that many "hyperplastic" parathyroid tumors in familial MEN-1 are in fact monoclonal and may progress or even begin to develop by inactivation of the MEN-1 gene (at 11q13) in a precursor cell. Some sporadic adenomas have allelic losses on chromosome 11, which may also involve the MEN-1 gene.

CCK-8 inhibits feeding-specific neurons in Navanax, an opisthobranch mollusc.

Cholecystokinin has been implicated as a satiety factor in mammals because it inhibits feeding through peripheral and central mechanisms. The cellular mechanisms of the central actions of CCK have been difficult to study because of the complex circuitry of the mammalian brain. Navanax is an opisthobranch mollusc with a defined neural network for feeding behavior in which the central effects of CCK can be studied at the cellular level. Here we report the localization of CCK-immunoreactivity in neuronal cell bodies and varicose fibers in the buccal ganglion of Navanax and that CCK-8 inhibits buccal ganglion neurons selectively and at tenth picomolar concentrations: expansion motoneurons responsible for prey capture are strongly inhibited by CCK-8; circumferential motoneurons responsible for swallowing are weakly inhibited by CCK-8. A large cell, cell X, is described which is sensitive to very low doses of CCK-8. These data imply the existence of a CCK-like peptide with transmitter-like actions in the buccal ganglion of Navanax.