SNCA genetic lowering reveals differential cognitive function of alpha-synuclein dependent on sex.

Antisense oligonucleotide (ASO) therapy for neurological disease has been successful in clinical settings and its potential has generated hope for Alzheimer's disease (AD). We previously described that ablating SNCA encoding for α-synuclein (αSyn) in a mouse model of AD was beneficial. Here, we sought to demonstrate whether transient reduction of αSyn expression using ASOSNCA could be therapeutic in a mouse model of AD. The efficacy of the ASOSNCA was measured via immunocytochemistry, RT-qPCR and western blotting. To assess spatial learning and memory, ASOSNCA or PBS-injected APP and non-transgenic (NTG) mice, and separate groups of SNCA-null mice, were tested on the Barnes circular maze. Hippocampal slice electrophysiology and transcriptomic profiling were used to explore synaptic function and differential gene expression between groups. Reduction of SNCA transcripts alleviated cognitive deficits in male transgenic animals, but surprisingly, not in females. To determine the functional cause of this differential effect, we assessed memory function in SNCA-null mice. Learning and memory were intact in male mice but impaired in female animals, revealing that the role of αSyn on cognitive function is sex-specific. Transcriptional analyses identified a differentially expressed gene network centered around EGR1, a central modulator of learning and memory, in the hippocampi of SNCA-null mice. Thus, these novel results demonstrate that the function of αSyn on memory differs between male and female brains.

Tau is required for progressive synaptic and memory deficits in a transgenic mouse model of α-synucleinopathy.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are clinically and neuropathologically highly related α-synucleinopathies that collectively constitute the second leading cause of neurodegenerative dementias. Genetic and neuropathological studies directly implicate α-synuclein (αS) abnormalities in PDD and DLB pathogenesis. However, it is currently unknown how αS abnormalities contribute to memory loss, particularly since forebrain neuronal loss in PDD and DLB is less severe than in Alzheimer's disease. Previously, we found that familial Parkinson's disease-linked human mutant A53T αS causes aberrant localization of the microtubule-associated protein tau to postsynaptic spines in neurons, leading to postsynaptic deficits. Thus, we directly tested if the synaptic and memory deficits in a mouse model of α-synucleinopathy (TgA53T) are mediated by tau. TgA53T mice exhibit progressive memory deficits associated with postsynaptic deficits in the absence of obvious neuropathological and neurodegenerative changes in the hippocampus. Significantly, removal of endogenous mouse tau expression in TgA53T mice (TgA53T/mTau-/-), achieved by mating TgA53T mice to mouse tau-knockout mice, completely ameliorates cognitive dysfunction and concurrent synaptic deficits without affecting αS expression or accumulation of selected toxic αS oligomers. Among the known tau-dependent effects, memory deficits in TgA53T mice were associated with hippocampal circuit remodeling linked to chronic network hyperexcitability. This remodeling was absent in TgA53T/mTau-/- mice, indicating that postsynaptic deficits, aberrant network hyperactivity, and memory deficits are mechanistically linked. Our results directly implicate tau as a mediator of specific human mutant A53T αS-mediated abnormalities related to deficits in hippocampal neurotransmission and suggest a mechanism for memory impairment that occurs as a consequence of synaptic dysfunction rather than synaptic or neuronal loss. We hypothesize that these initial synaptic deficits contribute to network hyperexcitability which, in turn, exacerbate cognitive dysfunction. Our results indicate that these synaptic changes present potential therapeutic targets for amelioration of memory deficits in α-synucleinopathies.

Bidirectional modulation of Alzheimer phenotype by alpha-synuclein in mice and primary neurons.

α-Synuclein (αSyn) histopathology defines several neurodegenerative disorders, including Parkinson's disease, Lewy body dementia, and Alzheimer's disease (AD). However, the functional link between soluble αSyn and disease etiology remains elusive, especially in AD. We, therefore, genetically targeted αSyn in APP transgenic mice modeling AD and mouse primary neurons. Our results demonstrate bidirectional modulation of behavioral deficits and pathophysiology by αSyn. Overexpression of human wild-type αSyn in APP animals markedly reduced amyloid deposition but, counter-intuitively, exacerbated deficits in spatial memory. It also increased extracellular amyloid-ß oligomers (AßOs), αSyn oligomers, exacerbated tau conformational and phosphorylation variants associated with AD, and enhanced neuronal cell cycle re-entry (CCR), a frequent prelude to neuron death in AD. Conversely, ablation of the SNCA gene encoding for αSyn in APP mice improved memory retention in spite of increased plaque burden. Reminiscent of the effect of MAPT ablation in APP mice, SNCA deletion prevented premature mortality. Moreover, the absence of αSyn decreased extracellular AßOs, ameliorated CCR, and rescued postsynaptic marker deficits. In summary, this complementary, bidirectional genetic approach implicates αSyn as an essential mediator of key phenotypes in AD and offers new functional insight into αSyn pathophysiology.

Discrete Pools of Oligomeric Amyloid-ß Track with Spatial Learning Deficits in a Mouse Model of Alzheimer Amyloidosis.

Despite increasing appreciation that oligomeric amyloid-ß (Aß) may contribute to cognitive decline of Alzheimer disease, defining the most critical forms has been thwarted by the changeable nature of these aggregates and the varying methods used for detection. Herein, using a broad approach, we quantified Aß oligomers during the evolution of cognitive deficits in an aggressive model of Aß amyloidosis. Amyloid precursor protein/tetracycline transactivator mice underwent behavioral testing at 3, 6, 9, and 12 months of age to evaluate spatial learning and memory, followed by histologic assessment of amyloid burden and biochemical characterization of oligomeric Aß species. Transgenic mice displayed progressive impairments in acquisition and immediate recall of the trained platform location. Biochemical analysis of cortical extracts from behaviorally tested mice revealed distinct age-dependent patterns of accumulation in multiple oligomeric species. Dot blot analysis demonstrated that nonfibrillar Aß oligomers were highly soluble and extracted into a fraction enriched for extracellular proteins, whereas prefibrillar species required high-detergent conditions to retrieve, consistent with membrane localization. Low-detergent extracts tested by 82E1 enzyme-linked immunosorbent assay confirmed the presence of bona fide Aß oligomers, whereas immunoprecipitation-Western blotting using high-detergent extracts revealed a variety of SDS-stable low-n species. These findings show that different Aß oligomers vary in solubility, consistent with distinct localization, and identify nonfibrillar Aß oligomer-positive aggregates as tracking most closely with cognitive decline in this model.

The amyloid-ß oligomer Aß*56 induces specific alterations in neuronal signaling that lead to tau phosphorylation and aggregation.

Oligomeric forms of amyloid-forming proteins are believed to be the principal initiating bioactive species in many neurodegenerative disorders, including Alzheimer's disease (AD). Amyloid-ß (Aß) oligomers are implicated in AD-associated phosphorylation and aggregation of the microtubule-associated protein tau. To investigate the specific molecular pathways activated by different assemblies, we isolated various forms of Aß from Tg2576 mice, which are a model for AD. We found that Aß*56, a 56-kDa oligomer that is detected before patients develop overt signs of AD, induced specific changes in neuronal signaling. In primary cortical neurons, Aß*56 interacted with N-methyl-d-aspartate receptors (NMDARs), increased NMDAR-dependent Ca2+ influx, and consequently increased intracellular calcium concentrations and the activation of Ca2+-dependent calmodulin kinase IIα (CaMKIIα). In cultured neurons and in the brains of Tg2576 mice, activated CaMKIIα was associated with increased site-specific phosphorylation and missorting of tau, both of which are associated with AD pathology. In contrast, exposure of cultured primary cortical neurons to other oligomeric Aß forms (dimers and trimers) did not trigger these effects. Our results indicate that distinct Aß assemblies activate neuronal signaling pathways in a selective manner and that dissecting the molecular events caused by each oligomer may inform more effective therapeutic strategies.

Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits.

Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-ß (Aß), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aß-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.

Soluble Conformers of Aß and Tau Alter Selective Proteins Governing Axonal Transport.

UNLABELLED: Despite the demonstration that amyloid-ß (Aß) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating Aß and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to Aß trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of Aß trimers in vivo, we created a novel bigenic Tg-Aß+Tau mouse line by crossing Tg2576 (Tg-Aß) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent Aß trimers were increased by ∼2-fold in 3-month-old Tg-Aß and Tg-Aß+Tau mice compared with younger mice, whereas soluble monomeric Aß levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ∼2.2-fold in the forebrains of Tg-Aß+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble Aß trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of Aß and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous Aß oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately. SIGNIFICANCE STATEMENT: The mechanistic link between amyloid-ß (Aß) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the trimeric Aß species induce a pathological modification of tau in cultured neurons and in bigenic mice expressing Aß and human tau. This linkage was also observed in postmortem brain tissue from subjects with mild cognitive impairment, when Aß trimers are abundant. Further, this modification of tau was associated with the intracellular accumulation of the precursor protein of Aß, APP, as a result of the selective decrease in kinesin light chain 1 expression. Our findings suggest that Aß trimers might cause axonal transport deficits in AD.

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive dementia disorder characterized by synaptic degeneration and amyloid-ß (Aß) accumulation in the brain. Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), we identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aß precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aß. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aß. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aß on synapses. In contrast, reduced PKCα activity is implicated in cancer. Hence, these findings reinforce the importance of maintaining a careful balance in the activity of this enzyme.

Genetic modulation of soluble Aß rescues cognitive and synaptic impairment in a mouse model of Alzheimer's disease.

An unresolved debate in Alzheimer's disease (AD) is whether amyloid plaques are pathogenic, causing overt physical disruption of neural circuits, or protective, sequestering soluble forms of amyloid-ß (Aß) that initiate synaptic damage and cognitive decline. Few animal models of AD have been capable of isolating the relative contribution made by soluble and insoluble forms of Aß to the behavioral symptoms and biochemical consequences of the disease. Here we use a controllable transgenic mouse model expressing a mutant form of amyloid precursor protein (APP) to distinguish the impact of soluble Aß from that of deposited amyloid on cognitive function and synaptic structure. Rapid inhibition of transgenic APP modulated the production of Aß without affecting pre-existing amyloid deposits and restored cognitive performance to the level of healthy controls in Morris water maze, radial arm water maze, and fear conditioning. Selective reduction of Aß with a γ-secretase inhibitor provided similar improvement, suggesting that transgene suppression restored cognition, at least in part by lowering Aß. Cognitive improvement coincided with reduced levels of synaptotoxic Aß oligomers, greater synaptic density surrounding amyloid plaques, and increased expression of presynaptic and postsynaptic markers. Together these findings indicate that transient Aß species underlie much of the cognitive and synaptic deficits observed in this model and demonstrate that significant functional and structural recovery can be attained without removing deposited amyloid.

Brain amyloid-ß oligomers in ageing and Alzheimer's disease.

Alzheimer's disease begins about two decades before the onset of symptoms or neuron death, and is believed to be caused by pathogenic amyloid-ß aggregates that initiate a cascade of molecular events culminating in widespread neurodegeneration. The microtubule binding protein tau may mediate the effects of amyloid-ß in this cascade. Amyloid plaques comprised of insoluble, fibrillar amyloid-ß aggregates are the most characteristic feature of Alzheimer's disease. However, the correspondence between the distribution of plaques and the pattern of neurodegeneration is tenuous. This discrepancy has stimulated the investigation of other amyloid-ß aggregates, including soluble amyloid-ß oligomers. Different soluble amyloid-ß oligomers have been studied in several mouse models, but not systematically in humans. Here, we measured three amyloid-ß oligomers previously described in mouse models-amyloid-ß trimers, Aß*56 and amyloid-ß dimers-in brain tissue from 75 cognitively intact individuals, ranging from young children to the elderly, and 58 impaired subjects with mild cognitive impairment or probable Alzheimer's disease. As in mouse models, where amyloid-ß trimers appear to be the fundamental amyloid-ß assembly unit of Aß*56 and are present in young mice prior to memory decline, amyloid-ß trimers in humans were present in children and adolescents; their levels rose gradually with age and were significantly above baseline in subjects in their 70s. Aß*56 levels were negligible in children and young adults, rose significantly above baseline in subjects in their 40s and increased steadily thereafter. Amyloid-ß dimers were undetectable until subjects were in their 60s; their levels then increased sharply and correlated with plaque load. Remarkably, in cognitively intact individuals we found strong positive correlations between Aß*56 and two pathological forms of soluble tau (tau-CP13 and tau-Alz50), and negative correlations between Aß*56 and two postsynaptic proteins (drebrin and fyn kinase), but none between amyloid-ß dimers or amyloid-ß trimers and tau or synaptic proteins. Comparing impaired with age-matched unimpaired subjects, we found the highest levels of amyloid-ß dimers, but the lowest levels of Aß*56 and amyloid-ß trimers, in subjects with probable Alzheimer's disease. In conclusion, in cognitively normal adults Aß*56 increased ahead of amyloid-ß dimers or amyloid-ß trimers, and pathological tau proteins and postsynaptic proteins correlated with Aß*56, but not amyloid-ß dimers or amyloid-ß trimers. We propose that Aß*56 may play a pathogenic role very early in the pathogenesis of Alzheimer's disease.

The complex PrP(c)-Fyn couples human oligomeric Aß with pathological tau changes in Alzheimer's disease.

Amid controversy, the cellular form of the prion protein PrP(c) has been proposed to mediate oligomeric amyloid-ß (Aß)-induced deficits. In contrast, there is consistent evidence that the Src kinase Fyn is activated by Aß oligomers and leads to synaptic and cognitive impairment in transgenic animals. However, the molecular mechanism by which soluble Aß activates Fyn remains unknown. Combining the use of human and transgenic mouse brain tissue as well as primary cortical neurons, we demonstrate that soluble Aß binds to PrP(c) at neuronal dendritic spines in vivo and in vitro where it forms a complex with Fyn, resulting in the activation of the kinase. Using the antibody 6D11 to prevent oligomeric Aß from binding to PrP(c), we abolished Fyn activation and Fyn-dependent tau hyperphosphorylation induced by endogenous oligomeric Aß in vitro. Finally, we showed that gene dosage of Prnp regulates Aß-induced Fyn/tau alterations. Together, our findings identify a complete signaling cascade linking one specific endogenous Aß oligomer, Fyn alteration, and tau hyperphosphorylation in cellular and animal models modeling aspects of the molecular pathogenesis of Alzheimer's disease.

Soluble α-synuclein is a novel modulator of Alzheimer's disease pathophysiology.

Recent evidence has emphasized soluble species of amyloid-ß (Aß) and tau as pathogenic effectors in Alzheimer's disease (AD). Despite the fact that Aß, tau, and α-synuclein (αSyn) can promote each other's aggregation, the potential contribution of soluble αSyn to AD pathogenesis is unknown. Here, we found an approximate twofold increase over controls in soluble αSyn levels in AD brains in the absence of Lewy body cytopathology. Importantly, soluble αSyn levels were a quantitatively stronger correlate of cognitive impairment than soluble Aß and tau levels. To examine a putative role for αSyn in modulating cognitive function, we used the Barnes circular maze to assess spatial reference memory in transgenic mice overexpressing human wild-type αSyn. The results revealed that an approximate threefold elevation of αSyn in vivo induced memory deficits similar to those observed in AD mouse models. The neurobiological changes associated with this elevation of soluble αSyn included decreases in selected synaptic vesicle proteins and an alteration of the protein composition of synaptic vesicles. Finally, a synergism between Aß/APP and human tau seems to be responsible for the abnormal elevation of soluble αSyn in transgenic mice. Altogether, our data reveal an unexpected role for soluble, intraneuronal αSyn in AD pathophysiology.

Detecting aß*56 oligomers in brain tissues.

Since its original description in 1906 by Dr Alois Alzheimer, amyloid plaques and neurofibrillary tangles have remained the hypothetical cause of Alzheimer's disease. However, plaque burden poorly predicts cognitive status in humans, which led several groups to investigate the possibility that soluble species of amyloid-beta (Aß) peptides could be playing an important pathological function in the aging brain. Through a multistep fractionation protocol, we identified a 56 kDa oligomer of Aß, termed Aß*56, the amount of which correlates with cognitive impairment. Here, we describe our biochemical approach to isolate this oligomeric Aß species in brain tissue of transgenic mouse models of AD.

Cognitive effects of cell-derived and synthetically derived Aß oligomers.

Soluble forms of amyloid-ß peptide (Aß) are a molecular focus in Alzheimer's disease research. Soluble Aß dimers (≈8 kDa), trimers (≈12 kDa), tetramers (≈16 kDa) and Aß*56 (≈56 kDa) have shown biological activity. These Aß molecules have been derived from diverse sources, including chemical synthesis, transfected cells, and mouse and human brain, leading to uncertainty about toxicity and potency. Herein, synthetic Aß peptide-derived oligomers, cell- and brain-derived low-n oligomers, and Aß*56, were injected intracerebroventricularly (icv) into rats assayed under the Alternating Lever Cyclic Ratio (ALCR) cognitive assay. Cognitive deficits were detected at 1.3 µM of synthetic Aß oligomers and at low nanomolar concentrations of cell-secreted Aß oligomers. Trimers, from transgenic mouse brain (Tg2576), did not cause cognitive impairment at any dose tested, whereas Aß*56 induced concentration-dependent cognitive impairment at 0.9 and 1.3µM. Thus, while multiple forms of Aß have cognition impairing activity, there are significant differences in effective concentration and potency.

Amyloid plaque and neurofibrillary tangle pathology in a regulatable mouse model of Alzheimer's disease.

Transgenic mouse models that independently express mutations in amyloid precursor protein (APP) and tau have proven useful for the study of the neurological consequences of amyloid-beta (Abeta) plaque and neurofibrillary tangle pathologies. Studies using these mice have yielded essential discoveries with regard to specific aspects of neuronal dysfunction and degeneration that characterize the brain during Alzheimer's disease (AD) and other age-dependent tauopathies. Most recent transgenic studies have focused on the creation of regulatable models that allow the temporal control of transgene expression. To study a more complete model of AD pathology, we designed a new regulatable transgenic mouse that harbors both APP and tau transgenes. Here, we present a novel transgenic mouse model, rTg3696AB, which expresses human APP(NLI) and tau(P301L) driven by the CaMKII promoter system. Subsequent generation of Abeta and 4R0N tau in the brain resulted in the development of three neuropathological features of AD: Abeta plaques, neurofibrillary tangles, and neurodegeneration. Importantly, transgene expression in these mice is regulatable, permitting temporal control of gene expression and the investigation of transgene suppression.

Involvement of beta-site APP cleaving enzyme 1 (BACE1) in amyloid precursor protein-mediated enhancement of memory and activity-dependent synaptic plasticity.

The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid-beta protein (Abeta), which forms amyloid plaques in Alzheimer's disease (AD), secreted APPalpha (sAPPalpha) which enhances memory, and the APP intracellular domain (AICD), which has been implicated in the regulation of gene transcription and calcium signaling. The beta-site APP cleaving enzyme 1 (BACE1) cleaves APP in an activity-dependent manner to form Abeta, AICD, and secreted APPbeta. Because this neural activity was shown to diminish synaptic transmission in vitro [Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R (2003) Neuron 37:925-937], the prevailing notion has been that this pathway diminishes synaptic function. Here we investigated the role of this pathway in vivo. We studied transgenic mice overproducing APP that do not develop AD pathology or memory deficits but instead exhibit enhanced spatial memory. We showed enhanced synaptic plasticity in the hippocampus that depends on prior synaptic activity. We found that the enhanced memory and synaptic plasticity are abolished by the ablation of one or both copies of the BACE1 gene, leading to a significant decrease in AICD but not of any other APP cleavage products. In contrast to the previously described negative effect of BACE1-mediated cleavage of APP on synaptic function in vitro, our in vivo work indicates that BACE1-mediated cleavage of APP can facilitate learning, memory, and synaptic plasticity.

Two phase I studies of low dose recombinant human IL-12 with Melan-A and influenza peptides in subjects with advanced malignant melanoma.

Preclinical studies have shown that low dose IL-12 can potentiate cytotoxic lymphocyte responses. Since previous trials have demonstrated significant toxicity from high dose recombinant human IL-12 (rhIL-12), we sought to determine an optimal biological dose for rhIL-12 at lower doses when combined with peptide antigens. Two studies were undertaken. The rhIL-12 was administered at doses of 0 (placebo), 10, 30 and 100 ng/kg, subcutaneously in one study and intravenously in the other. Apart from IL-12 dosing, the studies were identical. Subjects had evaluable stage III or IV melanoma which expressed Melan-A by RT-PCR or immunohistochemistry. Melan-A (26-35) (EAAGIGILTV) and influenza matrix (58-66) (GILGFVFTL) peptides were administered intradermally on weeks 1, 2, 3, 4 and 9. Twenty-eight subjects were enrolled, of whom 24 were evaluable for clinical and immunological responses. Therapy was well tolerated, the main adverse event being influenza-like symptoms. Immunological monitoring included the evaluation of cutaneous reactions and assays for antigen-specific T-cells. Clinical responses included a complete response in a subject with small volume subcutaneous disease, a partial response in a subject with hepatic metastases, and mixed responses in pulmonary, pleural and nodal disease. Biopsies of accessible tumors showed infiltration with CD4+ and CD8+ lymphocytes capable of lysing Melan-A peptide-pulsed targets in vitro. No clear dose-dependent effect of rhIL-12 could be determined. The rhIL-12 given either s.c. or i.v. was well tolerated at doses of 10-100 ng/kg. Clinical and immunological activity has been observed in this study where peptides were administered either with or without low dose rhIL-12.