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1.
Parkinsonism Relat Disord ; 87: 98-104, 2021 06.
Article in English | MEDLINE | ID: mdl-34020303

ABSTRACT

INTRODUCTION: Ubiquitous naturally occurring autoantibodies (nAbs) against alpha-synuclein (α-syn) may play important roles in the pathogenesis of Multiple System Atrophy (MSA) and Parkinson's disease (PD). Recently, we reported reduced high-affinity/avidity anti-α-syn nAbs levels in plasma from MSA and PD patients, along with distinct inter-group immunoglobulin (Ig)G subclass distributions. The extent to which these observations in plasma may reflect corresponding levels in the cerebrospinal fluid (CSF) is unknown. METHODS: Using competitive and indirect ELISAs, we investigated the affinity/avidity of CSF anti-α-syn nAbs as well as the CSF and plasma distribution of IgG subclasses and IgM nAbs in a cross-sectional cohort of MSA and PD patients. RESULTS: Repertoires of high-affinity/avidity anti-α-syn IgG nAbs were reduced in CSF samples from MSA and PD patients compared to controls. Furthermore, anti-α-syn IgM nAb levels were relatively lower in CSF and plasma from MSA patients but were reduced only in plasma from PD patients. Interestingly, anti-α-syn IgG subclasses presented disease-specific profiles both in CSF and plasma. Anti-α-syn IgG1, IgG2 and IgG3 levels were relatively increased in CSF of MSA patients, whereas PD patients showed increased anti-α-syn IgG2 and reduced anti-α-syn IgG4 levels. CONCLUSIONS: Differences in the plasma/CSF distribution of anti-α-syn nAbs seem to be a common feature of synucleinopathies. Our data add further support to the notion that MSA and PD patients may have compromised immune reactivity towards α-syn. The differing α-syn-specific systemic immunological responses may reflect their specific disease pathophysiologies. These results are encouraging for further investigation of these immunological mechanisms in neurodegenerative diseases.


Subject(s)
Autoantibodies , Multiple System Atrophy , Parkinson Disease , alpha-Synuclein/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Anti-Idiotypic/blood , Antibodies, Anti-Idiotypic/cerebrospinal fluid , Antibodies, Anti-Idiotypic/immunology , Autoantibodies/blood , Autoantibodies/cerebrospinal fluid , Autoantibodies/immunology , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Female , Humans , Male , Middle Aged , Multiple System Atrophy/blood , Multiple System Atrophy/cerebrospinal fluid , Multiple System Atrophy/immunology , Parkinson Disease/blood , Parkinson Disease/cerebrospinal fluid , Parkinson Disease/immunology
2.
Front Immunol ; 10: 2253, 2019.
Article in English | MEDLINE | ID: mdl-31616427

ABSTRACT

Aggregation of alpha-synuclein (α-syn) is considered to be the major pathological hallmark and driving force of Multiple System Atrophy (MSA) and Parkinson's disease (PD). Immune dysfunctions have been associated with both MSA and PD and recently we reported that the levels of natural occurring autoantibodies (NAbs) with high-affinity/avidity toward α-synuclein are reduced in MSA and PD patients. Here, we aimed to evaluate the plasma immunoglobulin (Ig) composition binding α-syn and other amyloidogenic neuropathological proteins, and to correlate them with disease severity and duration in MSA and PD patients. All participants were recruited from a single neurological unit and the plasma samples were stored for later research at the Bispebjerg Movement Disorder Biobank. All patients were diagnosed according to current consensus criteria. Using multiple variable linear regression analyses, we observed higher levels of anti-α-syn IgG1 and IgG3 NAbs in MSA vs. PD, higher levels of anti-α-syn IgG2 NAbs in PD compared to controls, whereas anti-α-syn IgG4 NAbs were reduced in PD compared to MSA and controls. Anti-α-syn IgM levels were decreased in both MSA and PD. Further our data supported that MSA patients' immune system was affected with reduced IgG1 and IgM global levels compared to PD and controls, with further reduced global IgG2 levels compared to PD. These results suggest distinct autoimmune patterns in MSA and PD. These findings suggest a specific autoimmune physiological mechanism involving responses toward α-syn, differing in neurodegenerative disease with overlapping α-syn pathology.


Subject(s)
Autoantibodies/immunology , Multiple System Atrophy/immunology , Parkinson Disease/immunology , alpha-Synuclein/immunology , Adult , Aged , Aged, 80 and over , Autoantibodies/blood , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Male , Middle Aged , Multiple System Atrophy/blood , Multiple System Atrophy/diagnosis , Parkinson Disease/blood , Parkinson Disease/diagnosis , Young Adult , alpha-Synuclein/blood
3.
Nat Immunol ; 19(11): 1212-1223, 2018 11.
Article in English | MEDLINE | ID: mdl-30323343

ABSTRACT

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.


Subject(s)
Antibodies, Monoclonal/immunology , Fibrinogen/antagonists & inhibitors , Neurodegenerative Diseases/immunology , Animals , Epitopes , Humans , Inflammation/immunology , Mice , Rats
4.
Mol Neurodegener ; 12(1): 44, 2017 06 07.
Article in English | MEDLINE | ID: mdl-28592329

ABSTRACT

BACKGROUND: Parkinson's' disease (PD) and Multiple System Atrophy (MSA) are progressive brain disorders characterized by intracellular accumulations of α-synuclein and nerve cell loss in specific brain areas. This loss causes problems with movement, balance and/or autonomic functions. Naturally occurring autoantibodies (NAbs) play potentially an important role in clearing or/and blocking circulating pathological proteins. Little is known about the functional properties of anti-α-synuclein NAbs in PD and MSA, and there have been opposing reports regarding their plasma concentrations in these disorders. METHODS: We have investigated the apparent affinity of anti-α-synuclein NAbs in plasma samples from 46 PD patients, 18 MSA patients and 41 controls using competitive enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) set-ups. RESULTS: We found that the occurrence of high affinity anti-α-synuclein NAbs in plasma from PD patients is reduced compared to healthy controls, and nearly absent in plasma from MSA patients. Also, levels of α-synuclein/NAbs immunocomplexes is substantially reduced in plasma from both patient groups. Further, cross binding of anti-α-synuclein NAbs with ß- and γ-synuclein monomers suggest, the high affinity anti-α-synuclein plasma component, seen in healthy individuals, is directed mainly against C-terminal epitopes. Furthermore, we also observed reduced occurrence of high affinity anti-phosphorylated-α-synuclein NAbs in plasma from PD and MSA patients. CONCLUSIONS: One interpretation implies that these patients may have impaired ability to clear and/or block the effects of pathological α-synuclein due to insufficient/absent concentration of NAbs and as such provides a rationale for testing immune-based therapeutic strategies directed against pathological α-synuclein. Following this interpretation, we can hypothesize that high affinity autoantibodies efficiently bind and clear potentially pathological species of α-synuclein in healthy brain, and that this mechanism is impaired or absent in PD and MSA patients.


Subject(s)
Autoantibodies/immunology , Multiple System Atrophy/immunology , Parkinson Disease/immunology , Aged , Brain/metabolism , Brain/pathology , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Male , Middle Aged , Multiple System Atrophy/pathology , Parkinson Disease/diagnosis , alpha-Synuclein/metabolism
5.
Biophys Chem ; 220: 34-41, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27863716

ABSTRACT

The 140-residue natively disordered protein α-synuclein (aSN) is a central component in the development of a family of neurodegenerative diseases termed synucleinopathies. This is attributed to its ability to form cytotoxic aggregates such as oligomers and amyloid fibrils. Consequently there have been intense efforts to avoid aggregation or reroute the aggregation pathway using pharmaceutical agents such as small molecules, chaperones and antibodies. aSN's lack of persistent structure in the monomeric state, as well as the multitude of different oligomeric and even different fibrillar states, makes it difficult to raise antibodies that would be efficacious in neutralizing all conformations of aSN. However, the C-terminal 20-40 residues of aSN are a promising epitope for antibody development. It is primarily disordered in both monomeric and aggregated forms, and an anti-C-terminal antibody will therefore be able to bind all forms. Furthermore, it might not interfere with the folding of aSN into membranes, which could be important for its physiological role. Here we report a screen of a series of monoclonal antibodies, which all target the C-terminal of aSN. According to dot blot analyses, different antibodies bound different forms of aSN with different preferences and showed reduced binding to monomeric compared to aggregated (oligomeric and fibrillary) aSN. Consequently they have different effects on aSN's ability to fibrillate and permeabilize membranes. Generally, the antibodies with strongest binding to aggregated aSN in dot blot, also inhibited fibrillation and membrane permeabilization the most, and promoted formation of amorphous aggregates surrounded by small and thin fibers. This suggests that the development of antibodies that targets the C-terminus, exposed in the aggregated forms of aSN, may be beneficial for improved immunotherapy against PD.


Subject(s)
Amyloid/drug effects , Antibodies, Monoclonal/pharmacology , Protein Aggregation, Pathological/prevention & control , alpha-Synuclein/immunology , Animals , Cell Membrane Permeability/drug effects , Humans , Mice , Parkinson Disease/drug therapy , Protein Aggregates/drug effects , Protein Aggregation, Pathological/drug therapy
6.
J Alzheimers Dis ; 52(2): 433-49, 2016 03 15.
Article in English | MEDLINE | ID: mdl-27003213

ABSTRACT

Aged companion dogs with canine cognitive dysfunction (CCD) spontaneously develop varying degrees of progressive cognitive decline and particular neuropathological features correspondent to the changes associated with Alzheimer's disease (AD) in humans. The aim of the present study was to characterize certain aspects of neuropathology and inflammatory markers related to aging and CCD in dogs in comparison with human AD. Fifteen brains from aged dogs with normal cognitive function, mild cognitive impairment, or CCD were investigated and compared with two control brains from young dogs and brain sections from human AD subjects. The neuropathological investigations included evaluation of amyloid-ß (Aß) plaque deposition (N-terminally truncated and pyroglutamyl-modified Aß included), tau pathology, and inflammatory markers in prefrontal cortex. Cortical Aß deposition was found to be only of the diffuse subtype as no dense-core or neuritic plaques were found. The Aß deposition followed a progressive pattern in four maturation stages. Accumulation of the Aß peptide was also observed in the vessel walls. Both immunohistochemically and biochemically measured levels of Aß pathology in prefrontal cortex showed a consistent positive correlation to age but not to cognitive deficit severity. No evidence of neurofibrillary tau pathology was found. The level of pro-inflammatory cytokines was generally low and showed no significant association to cognitive status. The findings of the present study support the senescent dog with spontaneous cognitive dysfunction as a valuable non-transgenic model for further investigations of the molecular events involved in the neurodegenerative processes associated with aging and early stage AD, especially the Aß-related pathology.


Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Disease Models, Animal , Dog Diseases , Aging/immunology , Aging/pathology , Aging/psychology , Alzheimer Disease/immunology , Alzheimer Disease/pathology , Animals , Brain/immunology , Brain/pathology , Cognitive Dysfunction/immunology , Cognitive Dysfunction/pathology , Cytokines/metabolism , Denmark/epidemiology , Disease Progression , Dog Diseases/immunology , Dog Diseases/pathology , Dogs , Female , Humans , Immunohistochemistry , Longitudinal Studies , Male , Plaque, Amyloid/immunology , Plaque, Amyloid/pathology , Severity of Illness Index , Species Specificity , Translational Research, Biomedical , tau Proteins/metabolism
7.
J Neuroimmunol ; 268(1-2): 50-7, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24507620

ABSTRACT

Immunotherapeutic approaches to treating Alzheimer's disease (AD) using vaccination strategies must overcome the obstacle of achieving adequate responses to vaccination in the elderly. Here we demonstrate for the first time that application of the Escherichia coli heat-labile enterotoxin adjuvant-laden immunostimulatory patches (LT-IS) dramatically enhances the onset and magnitude of immune responses to DNA- and protein-based vaccines for Alzheimer's disease following intradermal immunization via gene gun and conventional needles, respectively. Our studies suggest that the immune activation mediated by LT-IS offers improved potency for generating AD-specific vaccination responses that should be investigated as an adjuvant in the clinical arena.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Alzheimer Vaccines/immunology , Bacterial Toxins/administration & dosage , Enterotoxins/administration & dosage , Escherichia coli Proteins/administration & dosage , Vaccination/methods , Amyloid beta-Peptides/immunology , Animals , Antibodies, Viral/blood , Bacterial Toxins/immunology , Disease Models, Animal , Enterotoxins/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Escherichia coli , Escherichia coli Proteins/immunology , Female , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Transdermal Patch , Vaccines, DNA/immunology
8.
J Neurosci ; 33(11): 4923-34, 2013 Mar 13.
Article in English | MEDLINE | ID: mdl-23486963

ABSTRACT

The Alzheimer's disease (AD) process is understood to involve the accumulation of amyloid plaques and tau tangles in the brain. However, attempts at targeting the main culprits, neurotoxic Aß peptides, have thus far proven unsuccessful for improving cognitive function. Recent clinical trials with passively administrated anti-Aß antibodies failed to slow cognitive decline in mild to moderate AD patients, but suggest that an immunotherapeutic approach could be effective in patients with mild AD. Using an AD mouse model (Tg2576), we tested the immunogenicity (cellular and humoral immune responses) and efficacy (AD-like pathology) of clinical grade Lu AF20513 vaccine. We found that Lu AF20513 induces robust "non-self" T-cell responses and the production of anti-Aß antibodies that reduce AD-like pathology in the brains of Tg2576 mice without inducing microglial activation and enhancing astrocytosis or cerebral amyloid angiopathy. A single immunization with Lu AF20513 induced strong humoral immunity in mice with preexisting memory T-helper cells. In addition, Lu AF20513 induced strong humoral responses in guinea pigs and monkeys. These data support the translation of Lu AF20513 to the clinical setting with the aims of: (1) inducing therapeutically potent anti-Aß antibody responses in patients with mild AD, particularly if they have memory T-helper cells generated after immunizations with conventional tetanus toxoid vaccine, and (2) preventing pathological autoreactive T-cell responses.


Subject(s)
Alzheimer Disease/immunology , Alzheimer Disease/therapy , Amyloid beta-Peptides/chemistry , Epitopes, T-Lymphocyte/immunology , Peptide Fragments/chemistry , Vaccination/methods , Age Factors , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Peptides/immunology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Analysis of Variance , Animals , Antibodies, Anti-Idiotypic/blood , Antibodies, Anti-Idiotypic/pharmacology , Antibody Formation/immunology , Brain/metabolism , Brain/pathology , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Dose-Response Relationship, Immunologic , Enzyme-Linked Immunosorbent Assay , Epitopes, B-Lymphocyte/immunology , Epitopes, B-Lymphocyte/metabolism , Female , Guinea Pigs , Humans , Immunologic Memory/drug effects , Immunologic Memory/immunology , Macaca fascicularis , Male , Mice , Mice, Transgenic , Mutation/genetics , Neuroglia/drug effects , Neuroglia/immunology , Peptide Fragments/immunology , Peptide Fragments/metabolism , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Protein Binding/immunology , Surface Plasmon Resonance , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Vaccines/immunology
9.
J Neurochem ; 110(5): 1377-87, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19519664

ABSTRACT

Alzheimer's disease (AD) is hypothesized to result from elevated brain levels of beta-amyloid peptide (Abeta) which is the main component of plaques found in AD brains and which cause memory impairment in mice. Therefore, there has been a major focus on the development of inhibitors of the Abeta producing enzymes gamma-secretase and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1). In this study, we investigated the Abeta-lowering effects of the BACE1 inhibitor LY2434074 in vitro and in vivo, comparing it to the well characterized gamma-secretase inhibitor LY450139. We sampled interstitial fluid Abeta from awake APPswe/PS1dE9 AD mice by in vivo Abeta microdialysis. In addition, we measured levels of endogenous brain Abeta extracted from wildtype C57BL/6 mice. In our in vitro assays both compounds showed similar Abeta-lowering effects. However, while systemic administration of LY450139 resulted in transient reduction of Abeta in both in vivo models, we were unable to show any Abeta-lowering effect by systemic administration of the BACE1 inhibitor LY2434074 despite brain exposure exceeding the in vitro IC(50) value several fold. In contrast, significant reduction of 40-50% of interstitial fluid Abeta and wildtype cortical Abeta was observed when infusing LY2434074 directly into the brain by means of reverse microdialysis or by dosing the BACE1 inhibitor to p-glycoprotein (p-gp) mutant mice. The effects seen in p-gp mutant mice and subsequent data from our cell-based p-gp transport assay suggested that LY2434074 is a p-gp substrate. This may partly explain why BACE1 inhibition by LY2434074 has lower in vivo efficacy, with respect to decreased Abeta40 levels, compared with gamma-secretase inhibition by LY450139.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain/enzymology , Alanine/analogs & derivatives , Alanine/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Animals , Aspartic Acid Endopeptidases/metabolism , Azepines/pharmacology , Brain/drug effects , Cell Line , Cells, Cultured , Dogs , Dose-Response Relationship, Drug , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protease Inhibitors/pharmacology
10.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 62(Pt 12): 1179-84, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17142892

ABSTRACT

A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural peptide is LIMPARFYPK. The peptide does not make efficient use of the middle E pocket for binding, which leads to a rather superficial and exposed binding mode for the central peptide residues. Despite this, the peptide binds with high affinity (IC50 of 31 nM).


Subject(s)
DNA-Directed DNA Polymerase/chemistry , HLA-A Antigens/chemistry , Oligopeptides/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Crystallization , HLA-A11 Antigen , Hepatitis B virus/chemistry , Humans , Hydrogen Bonding , Protein Binding , Protein Conformation
11.
Int J Cancer ; 119(4): 831-8, 2006 Aug 15.
Article in English | MEDLINE | ID: mdl-16557580

ABSTRACT

Seminoma lesions are characterized by a brisk inflammatory infiltrate containing both CD4 and CD8 T cells, which is of prognostic significance. However, whether seminoma cells express the HLA molecules required for classical T-cell recognition remains controversial. In the present study, we conducted a molecular, phenotypical and functional characterization of tumor infiltrating lymphocytes (TILs) from seminoma lesions. T-cell receptor clonotype mapping demonstrated the presence of clonally expanded T cells in the majority of the lesions. The cytotoxic capacity of TILs was indicated by expression of CD107a, which is a recently described surrogate marker for cytolytic activity. Indeed, the frequency of CD107a positive cells was substantially higher in TILs when compared to peripheral blood mononuclear cells. Moreover, fluorescence activated cell sorting of CD107a positive TILs allowed comparison of the clonotypic T-cell receptor fingerprint and demonstrated the ability of expanded clones to express this cytotoxic marker, suggesting cytotoxic activity at the tumor site. The cytotoxicity was confirmed by in situ granzyme B expression. Furthermore, by staining with multimeric HLA-peptide complexes, we could demonstrate the presence of Mage-3 specific T cells among TILs. In summary, specific and functional T-cell responses are operative in seminoma, indicating that the inflammatory infiltrate is indeed involved in the immunological control of the tumor.


Subject(s)
Clone Cells/cytology , Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/immunology , Seminoma/immunology , Seminoma/pathology , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , Adult , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Clone Cells/immunology , Gene Expression Regulation, Neoplastic , Granzymes , Histocompatibility Antigens Class I/metabolism , Humans , Lymphocytes, Tumor-Infiltrating/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Middle Aged , Phenotype , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Seminoma/metabolism , Serine Endopeptidases/metabolism
12.
Cancer Immunol Immunother ; 55(5): 495-502, 2006 May.
Article in English | MEDLINE | ID: mdl-16001163

ABSTRACT

It is generally believed that priming of efficient T-cell responses takes place in peripheral lymphoid tissues. Although this notion has been rigidly proven for infectious diseases, direct evidence for lymph node priming of in vivo T-cell responses against tumors is still lacking. In the present study, we conducted a full and nonbiased comparison of T-cell clonotypes in melanoma lesions and corresponding sentinel lymph nodes. Whereas most tumor lesions comprised a high number of T-cell clonotypes, only a small number of clonally expanded T cells were detected in the draining lymph nodes. Comparative clonotype mapping demonstrated the presence of identical T-cell clonotypes in the tumors and the respective sentinel lymph nodes, only when tumor cells were present in the latter. However, taking advantage of clonotype specific PCR amplification, TCR sequences representing clonally expanded T cells at the tumor site could be detected in the lymph nodes draining the tumors even in the absence of tumor cells. Evidence for the tumor-specific characteristics of these cells was obtained by in situ staining with peptide/HLA class I complexes demonstrating the presence of MART-1/HLA-A2- and MAGE-3/HLA-A2-reactive T cells at the tumor site, as well as in the draining lymph node. Our data indicate that T-cell responses to melanoma are primed in the sentinel lymph node by cross presentation of tumor antigens by dendritic cells.


Subject(s)
Lymph Nodes/immunology , Lymphatic Metastasis/immunology , Melanoma/immunology , Receptors, Antigen, T-Cell/immunology , Skin Neoplasms/immunology , Adult , Aged , Antigens, Neoplasm , HLA Antigens/immunology , Humans , Immunohistochemistry , Lymph Nodes/pathology , Lymphatic Metastasis/pathology , Melanoma/pathology , Melanoma-Specific Antigens , Middle Aged , Neoplasm Proteins/immunology , Polymorphism, Single-Stranded Conformational , Reverse Transcriptase Polymerase Chain Reaction , Sentinel Lymph Node Biopsy , Skin Neoplasms/pathology
13.
Vaccine ; 23(7): 884-9, 2005 Jan 04.
Article in English | MEDLINE | ID: mdl-15603888

ABSTRACT

Prognosis of disseminated melanoma remains gloomy as neither chemotherapeutic nor unspecific immune modulatory approaches were able to improve the overall survival of these patients. Hence, specific immunotherapy has received increasing attention. Disappointing clinical results, however, indicate that the choice of suitable antigens is of special importance. To this end, the inhibitor of apoptosis (IAP) protein survivin, which is over-expressed in several tumours but is largely undetectable in adult tissues, appears to be a promising target for vaccination purposes, since down-regulation or loss of expression is associated with impaired tumour progression. Consequently, five heavily pretreated stage IV melanoma patients were vaccinated with the HLA-A2 restricted survivin(96-104) epitope presented by autologous dendritic cells (DCs) in a compassionate use setting. Four of these patients mounted strong T cell responses to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA-A2 multimer staining confirmed that these survivin reactive cells infiltrated both visceral and soft tissue metastases.


Subject(s)
Antigens, Neoplasm/therapeutic use , Cancer Vaccines/therapeutic use , Melanoma/therapy , Microtubule-Associated Proteins/therapeutic use , T-Lymphocytes/immunology , Adult , Aged , Antigens, Neoplasm/adverse effects , Antigens, Neoplasm/immunology , Cancer Vaccines/adverse effects , Cancer Vaccines/immunology , Female , HLA-A2 Antigen/immunology , Humans , Inhibitor of Apoptosis Proteins , Male , Melanoma/immunology , Microtubule-Associated Proteins/adverse effects , Microtubule-Associated Proteins/immunology , Middle Aged , Neoplasm Proteins , Survivin
14.
Int J Cancer ; 111(2): 229-37, 2004 Aug 20.
Article in English | MEDLINE | ID: mdl-15197776

ABSTRACT

Anticancer immune therapies aim at the induction of tumor-specific T cells, which ultimately should kill tumor cells. The effector cells should, therefore, not only exert cytotoxic activity but also home to and infiltrate the tumor site. Hence, monitoring of immune modulating therapies should not be restricted to the circulating pool of peripheral blood mononuclear cells (PBMC) but also include tumor-infiltrating lymphocytes (TIL), as well as the correlation of these findings to the clinical course. We report here on the longitudinal immunologic workup of a melanoma patient who developed remarkably potent ex vivo detectable antimelanoma cytotoxic T-cell (CTL) responses after vaccinations with autologous peptide-pulsed dendritic cells. Such potent CTL responses to multiple tumor antigens have, to the best of our knowledge, not been described previously in melanoma patients, neither spontaneously nor after any therapy. This patient first experienced a transient response to therapy but finally succumbed to disease progression and died. Progression was associated with the decline of the numbers of tumor-reactive T cells in circulation and at skin metastases in addition to the loss of MHC class I antigens. The immunologic analysis revealed that fully functional tumor-specific T cells were present in the peripheral blood of this patient during the phase of a relatively stable disease, and in situ tetramer staining demonstrated that these cells were also accumulated at cutaneous and visceral tumor sites. Furthermore, comparative clonotype mapping of PBMC and TIL depicted an overlapping TCR repertoire usage among these 2 compartments. Since strong CTL responses as observed in this patient are the goal of cancer vaccination but are so far only rarely observed, the thorough analysis of patients exhibiting either exceptional clinical and/or immunologic responses appears critical to understanding how vaccine therapies work and can be further improved.


Subject(s)
Cancer Vaccines , Dendritic Cells/immunology , Melanoma/immunology , Melanoma/pathology , Neoplastic Cells, Circulating , Skin Neoplasms/immunology , Skin Neoplasms/pathology , T-Lymphocytes, Cytotoxic/immunology , Aged , Antigens, Neoplasm/immunology , Base Sequence , Disease Progression , Fatal Outcome , Humans , Immunotherapy/methods , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating/immunology , Male , Melanoma/therapy , Molecular Sequence Data , Monocytes/immunology , Neoplasm Metastasis , Skin Neoplasms/therapy
15.
Arch Immunol Ther Exp (Warsz) ; 51(6): 377-88, 2003.
Article in English | MEDLINE | ID: mdl-14692659

ABSTRACT

The induction of a proper adaptive immune response is dependent on the correct transfer of information between antigen-presenting cells (APCs) and antigen-specific T cells. Defects in information transfer may result in the development of diseases, e.g. immunodeficiencies and autoimmunity. A distinct 3-dimensional supramolecular structure at the T cell/APC interface has been suggested to be involved in the information transfer. Due to its functional analogy to the neuronal synapse, the structure has been termed the "immunological synapse" (IS). Here, we review molecular aspects concerning IS formation, appearance, and cessation. In addition, proposed functions of the IS are discussed. The process of IS formation occurs in a sequential manner, initially causing a remarkable large-scale redistribution of a number of integral membrane and cytosolic proteins. At the T cell/APC interface the structure comprises in its nascent stage a non-random pattern of protein distribution. The protein pattern is regulated during development of the mature IS and is finally organized into concentric rings of co-receptors and adhesive molecules surrounding the T cell antigen receptor (TCR). The relocations of proteins are influenced by passive as well as active mechanisms. Considering the IS as a device enabling cell-cell communication, clarification of its exact function is of huge general as well as therapeutic interest.


Subject(s)
Antigen-Presenting Cells/immunology , Cell Communication/physiology , Immune System/physiology , T-Lymphocytes/immunology , Cell Adhesion , Cell Movement , Models, Immunological
17.
Proc Natl Acad Sci U S A ; 100(11): 6741-6, 2003 May 27.
Article in English | MEDLINE | ID: mdl-12746497

ABSTRACT

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood-brain barrier and provide neuroprotection.


Subject(s)
Brain Ischemia/drug therapy , Erythropoietin/pharmacology , Neuroprotective Agents/pharmacology , Animals , Erythropoietin/pharmacokinetics , Erythropoietin/therapeutic use , Hemoglobins/analysis , Male , Mice , Neuroprotective Agents/pharmacokinetics , Neuroprotective Agents/therapeutic use , Rats , Rats, Sprague-Dawley
18.
J Invest Dermatol ; 119(6): 1443-8, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12485452

ABSTRACT

Cellular immune responses are initiated by direct interaction of naive T cells with professional antigen-presenting cells, i.e., dendritic cells. In general, this interaction takes place in secondary lymphoid organs to which both naive T cells and mature dendritic cells preferentially home. This physiologic scenario differs substantially, however, from therapeutic dendritic-cell-based vaccinations used to treat human cancer. In fact, only a small fraction of intradermally injected dendritic cells migrate to the draining lymph node and the majority of cells remain at the site of inoculation. These sites are characterized by a distinct oligoclonal T cell infiltrate comprising both L-Selectin+/CD45RA+ and L-Selectin+/CD45RA- cells. Blood vessels expressing peripheral lymph node addressin represent possible entry channels for such naive and central memory T cells, the former probably attracted by dendritic cell-CK1 produced by the injected dendritic cells. In situ staining with multimeric peptide/major histocompatibility complex class I complexes revealed that infiltrating T cells specifically recognize peptide epitopes presented by the injected dendritic cells. Thus, the fraction of dendritic cells not migrating to secondary lymphatic tissue after therapeutic inoculation nevertheless seem to be involved in a specific immune modulation.


Subject(s)
Cell Aggregation/immunology , Dendritic Cells/cytology , T-Lymphocytes/cytology , Vaccination , Antigens, Neoplasm/immunology , Clone Cells , Dendritic Cells/immunology , Dermis/cytology , Dermis/immunology , Humans , Immunologic Memory , L-Selectin/analysis , Male , Melanoma/immunology , Receptors, Antigen, T-Cell/genetics , Skin Neoplasms/immunology , T-Lymphocytes/immunology
19.
J Invest Dermatol ; 118(4): 595-9, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11918704

ABSTRACT

Selection and activation of T cells is tightly regulated by both antigen-specific receptors and co-receptors to ensure that responses to self antigens are largely avoided. By T cell receptor clonotypic mapping and staining with tetrameric HLA-peptide complexes, we demonstrate the presence of melanocyte differentiation antigen MART-1 specific T cells in the areas of destruction of both neoplastic and normal melanocytic cells in a case of a primary melanoma and its associated hypopigmentation. These self reactive T cells expressed CD94/NKG2 major histocompatibility complex class I specific C-type lectin-like receptors. This family of receptors includes both activating and inhibitory isoforms. Thus, we performed a detailed analysis that revealed the exclusive presence of inhibitory NKG2-A/B receptors in the vitiligo-like leukoderma, whereas both the inhibitory receptors and the activating NKG2-C/E isoforms were present within the tumor. Our data suggest the differential expression of killer inhibitory receptors as a possible mechanism to regulate T cell responses to self antigens.


Subject(s)
Lectins, C-Type , Melanoma/immunology , Skin Neoplasms/immunology , T-Lymphocytes/immunology , Vitiligo/immunology , Antigens, CD/genetics , Antigens, Neoplasm , Autoimmunity/immunology , Clone Cells , Gene Expression Regulation, Neoplastic/immunology , Humans , Immune Tolerance/immunology , Immunohistochemistry , MART-1 Antigen , Membrane Glycoproteins/genetics , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , Neoplasm Proteins/analysis , Receptors, Antigen, T-Cell, alpha-beta/analysis , Receptors, Immunologic/genetics , Receptors, Natural Killer Cell , T-Lymphocytes/chemistry , T-Lymphocytes/cytology
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