Effects of single and combined immunotherapy approach targeting amyloid ß protein and α-synuclein in a dementia with Lewy bodies-like model.

INTRODUCTION: Immunotherapeutic approaches targeting amyloid ß (Aß) protein and tau in Alzheimer's disease and α-synuclein (α-syn) in Parkinson's disease are being developed for treating dementia with Lewy bodies. However, it is unknown if single or combined immunotherapies targeting Aß and/or α-syn may be effective. METHODS: Amyloid precursor protein/α-syn tg mice were immunized with AFFITOPEs® (AFF) peptides specific to Aß (AD02) or α-syn (PD-AFF1) and the combination. RESULTS: AD02 more effectively reduced Aß and pTau burden; however, the combination exhibited some additive effects. Both AD02 and PD-AFF1 effectively reduced α-syn, ameliorated degeneration of pyramidal neurons, and reduced neuroinflammation. PD-AFF1 more effectively ameliorated cholinergic and dopaminergic fiber loss; the combined immunization displayed additive effects. AD02 more effectively improved buried pellet test behavior, whereas PD-AFF1 more effectively improved horizontal beam test; the combined immunization displayed additive effects. DISCUSSION: Specific active immunotherapy targeting Aß and/or α-syn may be of potential interest for the treatment of dementia with Lewy bodies.

Evaluation of modified Interferon alpha mRNA constructs for the treatment of non-melanoma skin cancer.

Application of in vitro transcribed (IVT) messenger ribonucleic acid (mRNA) is an increasingly popular strategy to transiently produce proteins as therapeutics in a tissue or organ of choice. Here, we focused on the skin and aimed to test if whole human skin tissue explant technology can be used to evaluate the expression efficacy of different IVT Interferon alpha (IFN-α) mRNA constructs in situ, after biolistic delivery. Skin explants were viable and intact for at least five days based on histologic analysis and TUNEL staining. Using GFP reporter mRNA formulations, we found mostly epidermal expression after biolistic delivery. Two out of five sequence-optimized IFN-α mRNA variants resulted in significantly improved IFN-α protein expression in human skin compared to native IFN-α mRNA transfection. IFN-α secretion analysis of the surrounding culture media confirmed these results. We provide a proof-of-concept that IFN-α mRNA delivery into intact human full thickness skin explants can be utilized to test mRNA sequence modifications ex vivo. This approach could be used to develop novel mRNA-based treatments of common epidermal skin conditions including non-melanoma skin cancer, where IFN-α protein therapy has previously shown a strong therapeutic effect.

Active immunization against complement factor C5a: a new therapeutic approach for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by neuronal loss due to amyloid beta aggregations, neurofibrillary tangles, and prominent neuroinflammation. Recently, interference with neuroinflammation as a new therapeutic approach for AD treatment gained great interest. Microglia cells, one of the major contributors in neuroinflammation, are activated in response to misfolded proteins such as amyloid ß and cell debris leading to a sustained release of pro-inflammatory mediators. Especially, complement factor C5a and its receptor have been found to be up-regulated in microglia in the immediate surroundings of cerebral amyloid plaques and blocking of C5aR resulted in a reduction of pathological markers in a model of AD. Here, we investigate the effect of active vaccination against the complement factor C5a to interfere with neuroinflammation and neuropathologic alterations in a mouse model of AD. METHODS: Short antigenic peptides AFF1 and AFF2, which mimic a C-terminal epitope of C5a, were selected and formulated to vaccines. These vaccines are able to induce a highly specific antibody response to the target protein C5a. Tg2576 mice, a common model of AD, were immunized with these two C5a-peptide vaccines and the induced immune response toward C5a was analyzed by ELISA and Western blot analysis. The influence on memory retention was assessed by a contextual fear conditioning test. Microglia activation and amyloid plaque deposition in the brain was visualized by immunohistochemistry. RESULTS: Both C5a-targeting vaccines were highly immunogenic and induced sustained antibody titers against C5a. Tg2576 mice vaccinated at early stages of the disease showed significantly improved contextual memory accompanied by the reduction of microglia activation in the hippocampus and cerebral amyloid plaque load compared to control mice. Late-stage immunization also showed a decrease in the number of activated microglia, and improved memory function, however, had no influence on the amyloid ß load. CONCLUSION: C5a-peptide vaccines represent a safe and well-tolerated immunotherapy, which is able to induce a strong and specific immune response against the pro-inflammatory molecule C5a. In a mouse model of AD, C5a-peptide vaccines reduce microglia activation and thus neuroinflammation, which is supposed to lead to reduced neuronal dysfunction and AD symptomatic decline.

Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy.

BACKGROUND: Multiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia and dysautonomia. Histopathologically, the hallmark of MSA is the abnormal accumulation of alpha-synuclein (α-syn) within oligodendroglial cells, leading to neuroinflammation, demyelination and neuronal death. Currently, there is no disease-modifying treatment for MSA. In this sense, we have previously shown that next-generation active vaccination technology with short peptides, AFFITOPEs®, was effective in two transgenic models of synucleinopathies at reducing behavioral deficits, α-syn accumulation and inflammation. RESULTS: In this manuscript, we used the most effective AFFITOPE® (AFF 1) for immunizing MBP-α-syn transgenic mice, a model of MSA that expresses α-syn in oligodendrocytes. Vaccination with AFF 1 resulted in the production of specific anti-α-syn antibodies that crossed into the central nervous system and recognized α-syn aggregates within glial cells. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn, reduced demyelination in neocortex, striatum and corpus callosum, and reduced neurodegeneration. Clearance of α-syn involved activation of microglia and reduced spreading of α-syn to astroglial cells. CONCLUSIONS: This study further validates the efficacy of vaccination with AFFITOPEs® for ameliorating the neurodegenerative pathology in synucleinopathies.

Tailoring the antibody response to aggregated Aß using novel Alzheimer-vaccines.

Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aß1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study.

Pyroglutamylated amyloid-ß is associated with hyperphosphorylated tau and severity of Alzheimer's disease.

Pyroglutamylated amyloid-ß (pE(3)-Aß) has been suggested to play a major role in Alzheimer's disease (AD) pathogenesis as amyloid-ß (Aß) oligomers containing pE(3)-Aß might initiate tau-dependent cytotoxicity. We aimed to further elucidate the associations among pE(3)-Aß, full-length Aß and hyperphosphorylated tau (HP-τ) in human brain tissue. We examined 41 post mortem brains of both AD (n = 18) and controls. Sections from frontal and entorhinal cortices were stained with pE(3)-Aß, HP-τ and full-length Aß antibodies. The respective loads were assessed using image analysis and western blot analysis was performed in a subset of cases. All loads were significantly higher in AD, but when using Aß loads as independent variables only frontal pE(3)-Aß load predicted AD. In frontal and entorhinal cortices pE(3)-Aß load independently predicted HP-τ load while non-pE(3)-Aß failed to do so. All loads correlated with the severity of AD neuropathology. However, partial correlation analysis revealed respective correlations in the frontal cortex only for pE(3)-Aß load only while in the entorhinal cortex respective correlations were seen for both HP-τ and non-pE(3)-Aß loads. Mini Mental State Examination scores were independently predicted by entorhinal HP-τ load and by frontal pE(3)-Aß load. Here, we report an association between pE(3)-Aß and HP-τ in human brain tissue and an influence of frontal pE(3)-Aß on both the severity of AD neuropathology and clinical dementia. Our findings further support the notion that pE(3)-Aß may represent an important link between Aß and HP-τ, and investigations into its role as diagnostic and therapeutic target in AD are warranted.

Next-generation active immunization approach for synucleinopathies: implications for Parkinson's disease clinical trials.

Immunotherapeutic approaches are currently in the spotlight for their potential as disease-modifying treatments for neurodegenerative disorders. The discovery that α-synuclein (α-syn) can transmit from cell to cell in a prion-like fashion suggests that immunization might be a viable option for the treatment of synucleinopathies. This possibility has been bolstered by the development of next-generation active vaccination technology with short peptides-AFFITOPEs(®) (AFF)- that do not elicit an α-syn-specific T cell response. This approach allows for the production of long term, sustained, more specific, non-cross reacting antibodies suitable for the treatment of synucleinopathies, such as Parkinson's disease (PD). In this context, we screened a large library of peptides that mimic the C-terminus region of α-syn and discovered a novel set of AFF that identified α-syn oligomers. Next, the peptide that elicited the most specific response against α-syn (AFF 1) was selected for immunizing two different transgenic (tg) mouse models of PD and Dementia with Lewy bodies, the PDGF- and the mThy1-α-syn tg mice. Vaccination with AFF 1 resulted in high antibody titers in CSF and plasma, which crossed into the CNS and recognized α-syn aggregates. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn oligomers in axons and synapses, accompanied by reduced degeneration of TH fibers in the caudo-putamen nucleus and by improvements in motor and memory deficits in both in vivo models. Clearance of α-syn involved activation of microglia and increased anti-inflammatory cytokine expression, further supporting the efficacy of this novel active vaccination approach for synucleinopathies.

Distribution of the regulatory peptide alarin in the eye of various species.

Alarin is a recently discovered regulatory peptide with vasoconstrictive properties in murine skin. Control of vasoconstriction/-relaxation is essential for ocular blood flow and hence the eye's homeostasis, and regulatory peptides are involved in regulation of ocular blood flow. Here we describe the existence and distribution of alarin in the eye of human and potential experimental animals (rat, mouse). Eyes of rat, mouse, and human were prepared for immunohistochemistry against murine and human alarin, respectively. Additionally, double staining experiments for alarin and CD31 were performed in human choroidal flat-mount preparations. For documentation, confocal laser scanning microscopy was used while quantitative real-time-PCR was applied to confirm immunohistochemical data and to detect alarin mRNA expression in human retina and choroid. Alarin-like immunoreactivity (alarin-LI) was detected in corneal epi- and endothelium of human, mouse, and rat, as well as in the conjunctiva of mouse and rat. Alarin-LI was found in the iris of all the species investigated and, in humans, was concentrated around blood vessels. All three species showed distinctive alarin-LI in the non-pigmented epithelium of the ciliary body. In the retina of mouse and rat, maximum signals were detected in the outer nuclear and ganglion cell layer, whereas in humans a strong alarin-LI was found around retinal blood vessels and in intrinsic choroidal neurons (ICN). Quantitative RT-PCR in human confirmed alarin mRNA expression retina and choroid. The existence of alarin in cornea and conjunctiva might indicate a role in immune defense, while its presence in the non-pigmented ciliary epithelium favors an involvement in aqueous humor production. Alarin around blood vessels/in ICN might indicate an involvement in ocular blood flow regulation. Since alarin is found widely distributed in the eyes of species investigated, we were able to establish the basis for further functional experiments.

Distribution of alarin immunoreactivity in the mouse brain.

Alarin is a 25 amino acid peptide that belongs to the galanin peptide family. It is derived from the galanin-like peptide gene by a splice variant, which excludes exon 3. Alarin was first identified in gangliocytes of neuroblastic tumors and later shown to have a vasoactive function in the skin. Recently, alarin was demonstrated to stimulate food intake as well as the hypothalamic-pituitary-gonadal axis in rodents, suggesting that it might be a neuromodulatory peptide in the brain. However, the individual neurons in the central nervous system that express alarin have not been identified. Here, we determined the distribution of alarin-like immunoreactivity (alarin-LI) in the adult murine brain. The specificity of the antibody against alarin was demonstrated by the absence of labeling after pre-absorption of the antiserum with synthetic alarin peptide and in transgenic mouse brains lacking neurons expressing the GALP gene. Alarin-LI was observed in different areas of the murine brain. A high intensity of alarin-LI was detected in the accessory olfactory bulb, the medial preoptic area, the amygdala, different nuclei of the hypothalamus such as the arcuate nucleus and the ventromedial hypothalamic nucleus, the trigeminal complex, the locus coeruleus, the ventral chochlear nucleus, the facial nucleus, and the epithelial layer of the plexus choroideus. The distinct expression pattern of alarin in the adult mouse brain suggests potential functions in reproduction and metabolism.

Detection of peri-synaptic amyloid-ß pyroglutamate aggregates in early stages of Alzheimer's disease and in AßPP transgenic mice using a novel monoclonal antibody.

The neurodegenerative pathology in patients with Alzheimer's disease (AD) has been associated with the progressive accumulation of aggregated and post-translationally modified amyloid-ß (Aß) species. Among them, recent studies indicate that the pyroglutamate modification of Aß (pE(3)Aß) catalyzed by glutaminyl cyclase might play an important role in the pathogenesis of AD. Although the effects of the pyroglutamate modification on Aß aggregation and toxicity have been investigated, less is known about the distribution of pE(3)Aß across the spectrum of AD and in the brains of amyloid-ß protein precursor (AßPP) transgenic (tg) animals. For this purpose, we generated a novel monoclonal antibody (denominated D129) that specifically recognizes pE(3)Aß and characterized the patterns of distribution in the postmortem brain samples from AD patients divided by disease stage (Braak stage) and in AßPP tg mice. We found that in early stages of AD and young AßPP tg mice pE(3)Aß was found in discrete linear and granular aggregates in the neuropil that co-localized with the pre-synaptic protein synaptophysin and was in close opposition to dendrites labeled with MAP2. In later stages of AD and in older AßPP tg mice, pE(3)Aß was abundant in diffuse and mature plaques. In conclusion, this study suggests that peri-synaptic accumulation of pE(3)Aß might contribute to early cognitive dysfunction in AD.

Activation of chloride secretion via proteinase-activated receptor 2 in a human eccrine sweat gland cell line--NCL-SG3.

Proteinase-activated receptor 2 (PAR-2) has been shown to elicit secretion in a variety of secretory epithelial cells by the transepithelial movement of chloride ions across the apical membrane. However, it is not known whether these receptors are present and/or functional in the secretory epithelial cells of the human eccrine sweat gland. To investigate this possibility mRNA analysis, Ca2+ microspectrofluorimetry and the short circuit current (Isc) technique were used to quantify electrolyte transport in a cell line (NCL-SG3) derived from human eccrine sweat gland secretory epithelia. The results provided molecular and functional evidence of the presence of PAR-2 receptors in the NCL-SG3 cells and show that these receptors can activate transepithelial Cl- secretion possibly via Ca2+-activated Cl- channels.

Alarin is a vasoactive peptide.

Galanin-like peptide (GALP) is a hypothalamic neuropeptide belonging to the galanin family of peptides. The GALP gene is characterized by extensive differential splicing in a variety of murine tissues. One splice variant excludes exon 3 and results in a frame shift leading to a novel peptide sequence and a stop codon after 49 aa. In this peptide, which we termed alarin, the signal sequence of the GALP precursor peptide and the first 5 aa of the mature GALP are followed by 20 aa without homology to any other murine protein. Alarin mRNA was detected in murine brain, thymus, and skin. In accordance with its vascular localization, the peptide exhibited potent and dose-dependent vasoconstrictor and anti-edema activity in the cutaneous microvasculature, as was also observed with other members of the galanin peptide family. However, in contrast to galanin peptides in general, the physiological effects of alarin do not appear to be mediated via the known galanin receptors. Alarin adds another facet to the surprisingly high-functional redundancy of the galanin family of peptides.

Galanin-like peptides exert potent vasoactive functions in vivo.

The cutaneous vasculature plays a key role in the pathophysiology of inflammatory skin diseases. The vascular activity is under the control of the peripheral nervous system that includes locally released neuropeptides. Recently, we detected receptors for the neuropeptide galanin in association with dermal blood vessels, suggesting a role of the galanin-peptide-family in the regulation of the cutaneous microvasculature. Therefore, we have investigated galanin and galanin-like peptide (GALP), a neuropeptide previously only considered to be involved in metabolism and reproduction in the central nervous system, for vaso-modulatory activity in the murine skin in vivo. Picomole amounts of intradermally injected galanin and GALP decreased cutaneous blood flow and inhibited inflammatory edema formation. Both the full-length GALP (1-60) and the putative smaller proteolytic fragment GALP (3-32) showed similar effects. These activities are most likely mediated by galanin receptors galanin receptor subtype 2 (GalR2) and/or galanin receptor subtype 3 (GalR3), because reverse transcription-PCR analysis of murine skin revealed messenger RNA (mRNA) expression of GalR2 and GalR3 but not of galanin receptor subtype 1. The lack of galanin receptor mRNAs in endothelial and smooth muscle cells indicates a neuronal localization of these receptors around the vessels. These results indicate functional activity of GALP in the periphery in vivo and suggest a potential role as an inflammatory modulator.

Gangliocytes in neuroblastic tumors express alarin, a novel peptide derived by differential splicing of the galanin-like peptide gene.

In neuroblastic tumors a relationship of differentiation of the tumor to galanin receptor expression and antiproliferative and apoptotic effects upon activation of galanin receptors in neuroblastoma cells was reported. To elucidate the expression of other components of the galanin peptide family in neuroblastic tumors, RT-PCR analysis of a variety of human neuroblastic tumor tissues was performed. Ganglioneuroma tissues revealed the presence of a splice variant of the galanin-like peptide (GALP) mRNA, which results in exclusion of exon 3 and a frame shift after the signal peptide sequence of GALP. This generates a peptide of 25 amino acids, which we have termed alarin because of the N-terminal alanine and the C-terminal serine. The novel neuropeptide alarin does not reveal significant homology to other peptides. Immunohistochemistry with antibodies directed against synthetic alarin peptide detected specific cytoplasmic granular staining in ganglia of human ganglioneuroma and ganglioneuroblastoma, as well as differentiated tumor cells of neuroblastoma tissues. Undifferentiated neuroblasts of these tumor tissues did not show alarin-like immunoreactivity and alarin-specific mRNA. Our findings indicate that alarin expression is a feature of ganglionic differentiation in neuroblastic tumor tissues.

Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists.

The hypothalamic galanin-like peptide (GALP) was isolated by its ability to activate galanin receptors. The mature porcine GALP is a 60-amino acid neuropeptide proteolytically processed from a 120-amino acid precursor protein. It contains a region identical to the N-terminal 13-amino acids of the neuropeptide galanin. Within the sequence of human GALP (1-60) a potential proteolytic cleavage site between two basic amino acids is present at position 33, which might lead to a shorter C-terminally amidated peptide. In addition, the first two amino acids could be potentially removed via the action of dipeptidase IV. Ligand binding assays using the human neuroblastoma cell line SH-SY5Y transfected with the respective galanin receptors revealed that human GALP (1-60) displayed the highest affinity for the galanin receptor subtype GalR3 (IC50 = 10 nM) followed by GalR2 (IC50 = 28 nM) and GalR1 (IC50 = 77 nM). Ligand binding assays and functional studies showed that the human GALP (3-32) fragment was at least as potent as full length GALP (1-60). Other studies have shown that shorter fragments like human GALP (1-21) and GALP (22-60) were not effective on feeding responses in mice as compared to the full length peptide. Taken together these data suggest that the putative fragment GALP (3-32) might represent the strongest mediator of biological GALP activity. Furthermore it might be a useful tool to study the affinity of GALP to galanin receptors and to search for specific GALP receptors.

Galanin and galanin receptors in human cancers.

The increasing interest in peptides and peptide receptors in cancer is based on the possibility of receptor targeting, because peptide receptors are often expressed in different human tumors. The neuropeptide galanin has also been suggested to be involved in the development of neuroendocrine tumors based on the development of estrogen-induced tumors in estrogen-sensitive rats. This study summarizes our current knowledge on the expression of galanin peptide and galanin receptors in different human neuroendocrine tumors. The expression of both, peptide and corresponding receptor, seems to be a common feature of human gliomas, pheochromocytomas, pituitary and neuroblastic tumors. The co-expression of galanin and its receptors supports a role for galanin in tumor cell pathology via autocrine/paracrine mechanisms.

Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells.

Recently we have shown that galanin binding significantly correlates with survival in neuroblastoma patients, indicating a possible modulatory role of galanin receptors in neuroblastic tumor biology. However, the molecular mechanisms beyond this correlation have not been elucidated. Here, the cellular effects on activation of specific galanin receptor subtypes in human SH-SY5Y neuroblastoma cells were analyzed using a tetracycline-controlled expression system. Pharmacological studies confirmed the inducible expression of high affinity binding sites for galanin in SH-SY5Y cells transfected with the galanin receptors GalR1 (SY5Y/GalR1) and GalR2 (SY5Y/GalR2). Microphysiometry revealed that both receptor subtypes were able to mediate an intracellular signal upon galanin application. Interestingly, induction of receptor expression and treatment with 100 nm galanin resulted in a dramatic decrease in cell viability in SY5Y/GalR2 cells (93 +/- 3%) compared with a less pronounced effect in SY5Y/GalR1 cells (19 +/- 10%). The antiproliferative potency of galanin was 100-fold higher in SY5Y/GalR2 (50% effective concentration, 1.1 nm) than in SY5Y/GalR1 cells (50% effective concentration, 190 nm). Furthermore, activation of receptor expression and exposure to galanin resulted in apparent morphological changes indicative of apoptosis in SY5Y/GalR2 cells only. Induction of cell death by the apoptotic process was confirmed by poly-(ADP-ribose)-polymerase cleavage, caspase-3 activation, and the typical laddering of DNA. This study indicates that a high level of GalR2 expression is able to inhibit cell proliferation and induce apoptosis in neuroblastoma cells and therefore identifies GalR2 as a possible target for pharmacological intervention in neuroblastoma.

Galanin and galanin receptors in human gliomas.

Galanin-like immunoreactivity (GAL-LI) and specific GAL binding sites have been shown to be widely distributed in the central nervous system (CNS) and in CNS tumors. GAL and its receptors have also been shown to be present in glial cells, but to date it is still unknown whether human gliomas produce GAL and express GAL receptors. In this study 20 brain tumors consisting of 15 glioblastomas, 4 meningiomas and 1 gliosarcoma were investigated for the presence of GAL-LI and GAL receptors. Immunofluorescence analysis revealed a dense network of GAL-LI positive cellular processes and cell bodies in 18 of the 20 tumors. In contrast, in vitro (125)I-labeled GAL receptor autoradiography showed substantial GAL binding in only 6 glioblastoma tissues. Reverse transcription-PCR analysis detected mRNA of all three known galanin receptors in the tumor tissues, with most tumors expressing multiple receptor subtypes. Pharmacological analysis of tumor membrane homogenates with GAL and the specific GAL receptor GalR2 agonist, AR-M1896, revealed that the GAL receptor GalR1 is most likely the receptor responsible for the observed GAL binding in the glioblastomas. No correlation could be found between GAL-LI, the level of GAL binding and proliferative activity as determined by immunostaining with the cell proliferation marker Ki-67.

125I-labeled galanin binding sites in congenital innervation defects of the distal colon.

Neuropeptides have turned out to be promising new parameters, in addition to the routinely performed histochemical diagnosis, of Hirschsprung's disease (HD). Studies of the peptidergic innervation of the affected intestinal segment of patients with HD have demonstrated a marked reduction in the density of several neuropeptide-containing nerve fibers. The frequency of nerve fibers storing the neuropeptide galanin (GAL) was found to be unchanged or slightly reduced in HD, but nothing is known about the occurrence of GAL receptors. In this study, in vitro receptor autoradiography using (125)I-labeled GAL and GAL immunofluorescence have been performed on frozen tissue sections from colon biopsies of 10 patients diagnosed with HD, 8 patients with intestinal neuronal dysplasia (IND B) and 20 patients with chronic obstruction but normal innervation. Binding sites were mainly detected in the mucosal and muscular layer, in acetylcholinesterase-positive nerve fiber bundles and ganglia within the submucosal layer and in close association to blood vessels. An increased population of GAL receptor positive, parasympathetic nerve fibers was seen in the aganglionic segment of HD as compared to controls and IND B. In contrast, GAL immunostaining which was unchanged in HD revealed a significant lack of GAL-positive structures in IND B colon biopsies. Colocalization of GAL and GAL binding sites was only observed in thick nerve fibers in the submucosa. The presence of GAL binding sites in different cellular structures suggests an involvement of GAL in various physiological functions of the gastrointestinal tract.