Multiple endocrine neoplasia 2A due to a unique C609S RET mutation presents with pheochromocytoma and reduced penetrance of medullary thyroid carcinoma.

OBJECTIVE: We have identified a large kindred with multiple endocrine neoplasia 2A (MEN 2A) due to a mutation at RET codon 609 that results in a cysteine to serine substitution, a mutation previously identified in only one case in the literature. We characterized the clinical phenotype of the kindred and the biochemical mechanism of this new mutation. PATIENTS AND DESIGN: The index case, a 42-year-old woman, presented with pheochromocytoma. We screened 29 family members for the presence of the mutation. Of the 15 mutation-positive family members, 11 agreed to undergo further evaluation by physical examination, calcium and pentagastrin-stimulated calcitonin levels, measurement of urinary metanephrines, adrenal imaging and serum calcium levels. Biochemical characterization of the mutation was by transient transfection of human neuroblastoma cells and Western blot analysis. RESULTS: This kindred demonstrated an inheritance pattern consistent with autosomal dominant pheochromocytoma. Strikingly, no clinically evident case of medullary thyroid cancer (MTC) was observed among mutation-positive family members. Thyroidectomy in six cases revealed C-cell hyperplasia in all and microscopic MTC in two cases. Transfection experiments using human neuroblastoma cells showed that the mutant RET, unlike the wild-type receptor, is constitutively phosphorylated in the absence of ligand, and thus resembles other previously characterized MEN 2A mutations. CONCLUSIONS: The identification of a new mutation causing a MEN 2A phenotype that features pheochromocytoma and the surprising absence of clinically apparent MTC has significant implications for carriers of this mutation and provides further insights into the genotype-phenotype correlation in MEN 2A.

Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo.

One of the most clinically advanced forms of experimental disease-modifying treatment for Alzheimer disease is immunization against the amyloid beta protein (Abeta), but how this may prevent cognitive impairment is unclear. We hypothesized that antibodies to Abeta could exert a beneficial action by directly neutralizing potentially synaptotoxic soluble Abeta species in the brain. Intracerebroventricular injection of naturally secreted human Abeta inhibited long-term potentiation (LTP), a correlate of learning and memory, in rat hippocampus in vivo but a monoclonal antibody to Abeta completely prevented the inhibition of LTP when injected after Abeta. Size fractionation showed that Abeta oligomers, not monomers or fibrils, were responsible for inhibiting LTP, and an Abeta antibody again prevented such inhibition. Active immunization against Abeta was partially effective, and the effects correlated positively with levels of antibodies to Abeta oligomers. The ability of exogenous and endogenous antibodies to rapidly neutralize soluble Abeta oligomers that disrupt synaptic plasticity in vivo suggests that treatment with such antibodies might show reversible cognitive deficits in early Alzheimer disease.

Differences in the immune response to long term Abeta vaccination in C57BL/6 and B6D2F1 mice.

The cerebral accumulation of beta-amyloid (Abeta) is a pathological hallmark of Alzheimer's disease (AD). Abeta vaccination or anti-Abeta specific antibodies may be a possible therapeutic option for AD. Previously, we demonstrated variation in the humoral response between B6D2F1 and C57BL/6 during short term (14 weeks) Abeta immunization. In the present study, we determined the humoral and cellular immune responses in these same mouse strains to a longer period of Abeta vaccination and further refined the major B cell epitope to Ass1-7. B6D2F1 mice generated a greater humoral and Th1 immune response versus C57BL/6 mice. Immunization with 25 microg Abeta produced a greater T cell response in B6D2F1 mice compared to 50 or 100 microg Abeta but resulted in comparable humoral immunity. Thus, Abeta vaccination is affected by the genetic background and amount of Abeta peptide used as immunogen. These data may help explain some differences observed in Abeta immunization studies in mice of various genetic backgrounds and aid in the design of Abeta vaccines.

Species-specific immune response to immunization with human versus rodent A beta peptide.

Amyloid beta (A beta) immunization of amyloid precursor protein (APP)-transgenic (tg) mice with human A beta induces humoral immunity, however, the immune response to endogenous rodent A beta is unknown. Fourteen-month J20 APP-tg mice and non-tg littermates were immunized subcutaneously followed by chronic intranasal boosting with human or rodent A beta peptide and adjuvant LT(R192G). Rodent A beta-immunized APP-tg mice had anti-rodent A beta antibody levels of 257.8 micrograms/ml and those immunized with human A beta had anti-human A beta antibodies of 120.8 micrograms/ml. Non-tg littermates had anti-rodent and anti-human A beta antibody concentrations of 98.8 and 231.1 microgram/ml, respectively. Inter-species cross-reactivity was minimal. Anti-human A beta antibodies were predominately IgG1 and IgG2b, while anti-rodent A beta antibodies were equally IgG1, IgG2a, and IgG2b. Anti-human A beta antibodies recognized an epitope within human A beta1-9. Anti-rodent A beta antibodies did not stain Alzheimer's disease (AD) plaques but bound some plaques in APP-tg mice. Splenocytes proliferated modestly to their respective antigen and secreted low levels of IL-2 and IFN-gamma. Therefore, immunizing APP-tg and non-tg mice with rodent A beta resulted in a species-specific humoral response with modest T cell reactivity.

Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet.

Amyloid beta (Abeta) protein immunotherapy lowers cerebral Abeta and improves cognition in mouse models of Alzheimer's disease (AD). Here we show that Caribbean vervet monkeys (Chlorocebus aethiops, SK) develop cerebral Abeta plaques with aging and that these deposits are associated with gliosis and neuritic dystrophy. Five aged vervets were immunized with Abeta peptide over 10 months. Plasma and cerebral spinal fluid (CSF) samples were collected periodically from the immunized vervets and five aged controls; one monkey per group expired during the study. By Day 42, immunized animals generated plasma Abeta antibodies that labeled Abeta plaques in human, AD transgenic mouse and vervet brains; bound Abeta1-7; and recognized monomeric and oligomeric Abeta but not full-length amyloid precursor protein nor its C-terminal fragments. Low anti-Abeta titers were detected in CSF. Abetax-40 levels were elevated approximately 2- to 5-fold in plasma and decreased up to 64% in CSF in immunized vervets. Insoluble Abetax-42 was decreased by 66% in brain homogenates of the four immunized animals compared to archival tissues from 13 age-matched control vervets. Abeta42-immunoreactive plaques were detected in frontal cortex in 11 of the 13 control animals, but not in six brain regions examined in each of the four immunized vervets. No T cell response or inflammation was observed. Our study is the first to demonstrate age-related Abeta deposition in the vervet monkey as well as the lowering of cerebral Abeta by Abeta vaccination in a non-human primate. The findings further support Abeta immunotherapy as a potential prevention and treatment of AD.

Evidence for peripheral clearance of cerebral Abeta protein following chronic, active Abeta immunization in PSAPP mice.

Immunization with amyloid-beta (Abeta) peptide in mouse models of Alzheimer's disease has been reported to decrease cerebral Abeta levels and improve behavioral deficits. Several mechanisms have been proposed, including antibody-induced phagocytosis of Abeta by cerebral microglia and increased efflux of Abeta from the brain to the periphery. The latter mechanism was suggested in mice undergoing acute, passive transfer of an Abeta monoclonal antibody. Here, PSAPP transgenic mice were actively immunized by a single intraperitoneal injection of synthetic Abeta followed by chronic intranasal administration of Abeta with the mucosal adjuvant, Escherichia coli heat-labile enterotoxin, LT, twice weekly for 8 weeks. Serum from Abeta-immunized mice had an average of 240 microg/ml of anti-Abeta-specific antibodies; these antibodies had epitope(s) within Abeta1-15 and were of immunoglobulin (Ig) isotypes IgG2b, IgG2a, and IgG1. Immunization led to a 75% decrease in plaque number (P < 0.0001) and a 58% decrease in Abetax-42 levels (P < 0.026) in brain, and gliosis and neuritic dystrophy were diminished. No pathological effects of the immunization were observed in kidney, spleen, or snout. Serum Abeta levels increased 28-fold in immunized mice (53.06 ng/ml) compared to controls (1.87 ng/ml). Most of the Abeta in the serum of the immunized mice was bound to antibodies. We conclude that following active immunization, anti-Abeta antibodies sequester serum Abeta and may increase central nervous system to serum Abeta clearance.

Amyloid-beta immunization in Alzheimer's disease transgenic mouse models and wildtype mice.

Alzheimer's disease is the most prevalent form of dementia worldwide. Therapies are desperately needed to prevent and cure the disease. Mouse models of amyloid-beta deposition [APP and PSAPP transgenic (tg) mice] have been useful in determining the role of amyloid-beta (A beta) in both the pathogenesis and cognitive changes in AD. In addition, they have allowed scientists to investigate potential AD therapies in living animals. Active and passive A beta immunizations have been employed successfully in APP and PSAPP tg mice to lower cerebral A beta levels and improve cognition. Optimization of immunization protocols and characterization of immune responses in wildtype mice have been reported. Based on the promising results of A beta immunization studies in mice, a clinical trial was initiated for A beta vaccination in humans with AD. Although no adverse effects were reported in the Phase I safety trials, about 5% of AD patients in the phase II clinical trial developed meningoencephalitis, ending the trial prematurely in March 2002. Studies in AD mouse models and wildtype mice may help elucidate the mechanism for these unwanted side effects and will be useful for testing newer, safer vaccines for future use in human clinical trials.

Abeta1-15 is less immunogenic than Abeta1-40/42 for intranasal immunization of wild-type mice but may be effective for "boosting".

Immunizing mouse models of Alzheimer's disease (AD) against beta-amyloid (Abeta) leads to a decrease in cerebral Abeta burden as well as an improvement in behavioral deficits. Circulating Abeta-antibodies may be responsible for interfering with Abeta deposition. In the present study, we attempted to initiate more robust antibody production in wild type (WT) mice. Three immunization strategies were examined: intranasal (i.n.) immunization with Abetal-15 or full-length Abeta1-40/42, i.n. administration of Abeta combined with mucosal adjuvants, native labile enterotoxin (LT) or its non-toxic form, LT(R192G), and prime-boost regimes. Using Abeta1-15 as the primary immunogen for intranasal immunization did not initiate strong antibody production. When Abeta1-15 or Abeta1-40/42 was combined with native LT or LT(R192G), antibody production was significantly increased. Nasal immunization with Abeta1-15 and native LT successfully "boosted" an immune response "primed" by an intraperitoneal (i.p.) injection of Abeta1-40/42, producing moderately high Abeta titers that remained stable for at least 6 months. Serum anti-Abeta antibodies, regardless of the length of the Abeta immunogen, consistently detected human AD plaques, had epitopes within Abeta1-15, and were predominantly of the IgG2b, IgG1, and IgG2a isotypes. The adjuvants were well-tolerated in the mice. Thus, Abeta1-15 may have potential as a safer, more cost-effective "boosting" immunogen than the full-length Abeta peptide for chronic, active Abeta immunization.

Intranasal immunotherapy for the treatment of Alzheimer's disease: Escherichia coli LT and LT(R192G) as mucosal adjuvants.

Alzheimer's disease (AD) is the most common form of dementia worldwide, yet there is currently no effective treatment or cure. Extracellular deposition of amyloid-beta protein (Abeta) in brain is a key neuropathological characteristic of AD. In 1999, Schenk et al. first reported that an injected Abeta vaccine given to PDAPP mice, an AD mouse model displaying Abeta deposition in brain, led to the lowering of Abeta levels in brain. In 2000, we demonstrated that intranasal (i.n.) immunization with human synthetic Abeta1-40 peptide for 7 months led to a 50-60% reduction in cerebral Abeta burden in PDAPP mice; serum Abeta antibody titers were low (approximately 26 microg/ml). More recently, we have optimized our i.n. Abeta immunization protocol in wild-type (WT) mice. When low doses Escherichia coli heat-labile enterotoxin (LT) were given as a mucosal adjuvant with Abeta i.n., there was a dramatic 12-fold increase in Abeta antibody titers in WT B6D2F1 mice treated two times per week for 8 weeks compared to those of mice receiving i.n. Abeta without adjuvant. A non-toxic form of LT, designated LT(R192G), showed even better adjuvanticity; anti-Abeta antibody titers were 16-fold higher than those seen in mice given i.n. Abeta without adjuvant. In both cases, the serum Abeta antibodies recognized epitopes within Abeta1-15 and were of the immunoglobulin (Ig) isotypes IgG2b, IgG1, IgG2a and low levels of IgA. This new and improved Abeta vaccine protocol is now being tested in AD mouse models with the expectation that higher Abeta antibody titers may be more effective in reducing cerebral Abeta levels.

Intraneuronal Abeta42 accumulation in Down syndrome brain.

Alzheimer's disease (AD) brains display A beta (Abeta) plaques, inflammatory changes and neurofibrillary tangles (NFTs). Converging evidence suggests a neuronal origin of Abeta. We performed a temporal study of intraneuronal Abeta accumulation in Down syndrome (DS) brains. Sections from temporal cortex of 70 DS cases aged 3 to 73 years were examined immunohistochemicallyf or immunoreactivity (IR) for the Abeta N-terminal, the Abeta40 C-terminus and the Abeta42 C-terminus. N-terminal antibodies did not detect intracellular Abeta. Abeta40 antibodies did not detect significant intracellular Abeta, but older cases showed Abeta40 IR in mature plaques. In contrast, Abeta42 antibodies revealed clear-cut intraneuronal IR. All Abeta42 antibodies tested showed strong intraneuronal Abeta42 IR in very young DS patients, especially in theyoungest cases studied (e.g., 3 or 4yr. old), but this IR declined as extracellular Abeta plaques gradually accumulated and matured. No inflammatory changes were associated with intraneuronal Abeta. We also studied the temporal development of gliosis and NFT formation, revealing that in DS temporal cortex, inflammation and NFT follow Abeta deposition. We conclude that Abeta42 accumulates intracellularly prior to extracellular Abeta deposition in Down syndrome, and that subsequent maturation of extracellular Abeta deposits elicits inflammatory responses andprecedes NFTs.

The generation and characterization of potentially therapeutic Abeta antibodies in mice: differences according to strain and immunization protocol.

Previous studies have shown that in various mouse models of Alzheimer's disease (AD), amyloid beta-protein (Abeta) antibodies generated by Abeta peptide immunization resulted in the prevention of Abeta plaque formation in brains of young mice, decreased Abeta plaque burdens in older mice and improved cognition. The purpose of this study was to optimize Abeta immunization protocols for future trials in transgenic mouse models of AD. The timing and titers of Abeta antibody production, as well as epitope(s) and imunoglobulin isotypes, were compared between two different mouse strains (C57BL/6 and B6D2F1) and five treatment protocols: (1). chronic Abeta nasal administration, (2). repeated Abeta intraperitoneal (i.p.) injection, (3). one i.p. injection followed by chronic Abeta nasal administration, (4). chronic and concurrent Abeta nasal administration + Abeta i.p. injection, and (5). untreated controls. B6D2F1 mice generated Abeta antibodies earlier and in higher quantities than the C57BL/6 mice, indicating that B6D2F1 mice are more responsive to Abeta immunization. For both strains, mice that received the combination of Abeta nasal + Abeta i.p. injection showed the highest antibody titers. Epitope mapping experiments indicated that the mouse anti-Abeta antibodies recognize residues within Abeta1-15. Immunoglobulin isotyping demonstrated that the Abeta antibodies are of the Th-2 anti-inflammatory type, IgG1 and IgG2b, with a few IgM. Currently there is no effective therapy for Alzheimer's disease; thus if Abeta immunization proves effective, it would be a significant step in the prevention and/or treatment of this devastating disease.