One gene but different proteins and diseases: the complexity of dystonin and bullous pemphigoid antigen 1.

Since the immunochemical identification of the bullous pemphigoid antigen 230 (BP230) as one of the major target autoantigens of bullous pemphigoid (BP) in 1981, our understanding of this protein has significantly increased. Cloning of its gene, development and characterization of animal models with engineered gene mutations or spontaneous mouse mutations have revealed an unexpected complexity of the gene encoding BP230. The latter, now called dystonin (DST), is composed of at least 100 exons and gives rise to three major isoforms, an epithelial, a neuronal and a muscular isoform, named BPAG1e (corresponding to the original BP230), BPAG1a and BPAG1b, respectively. The various BPAG1 isoforms play a key role in fundamental processes, such as cell adhesion, cytoskeleton organization, and cell migration. Genetic defects of BPAG1 isoforms are the culprits of epidermolysis bullosa and complex, devastating neurological diseases. In this review, we summarize recent advances of our knowledge about several BPAG1 isoforms, their role in various biological processes and in human diseases.

Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

A significant effect of the KIR ligand HLA-C on fibrosis progression in chronic C hepatitis with or without liver transplantation.

BACKGROUND & AIMS The interaction of KIR with their HLA ligands drives the activation and inhibition of natural killer (NK) cells. NK cells could be implicated in the development of liver fibrosis in chronic hepatitis C. METHODS We analysed 206 non-transplanted and 53 liver transplanted patients, selected according to their Metavir fibrosis stage. Several variables such as the number of activator KIR or the HLA ligands were considered in multinomial and logistic regression models. Possible confounding variables were also investigated. RESULTS The KIRs were not significant predictors of the fibrosis stage. Conversely, a significant reduction of the HLA-C1C2 genotype was observed in the most advanced fibrosis stage group (F4) in both cohorts. Furthermore, the progression rate of fibrosis was almost 10 times faster in the subgroup of patients after liver transplantation and HLA-C1C2 was significantly reduced in this cohort compared to non-transplanted patients. CONCLUSION This study suggests a possible role of KIR and their ligands in the development of liver damage. The absence of C1 and C2 ligands heterozygosity could lead to less inhibition of NK cells and a quicker progression to a high level of fibrosis in patients infected by HCV, especially following liver transplantation. This article is protected by copyright. All rights reserved.

HLA-A29-POSITIVE BIRDSHOT CHORIORETINOPATHY IN AN AFRICAN AMERICAN PATIENT.

PURPOSE: To report the first documented case of HLA-A29-positive birdshot chorioretinopathy in an African American patient. METHODS: A 51-year-old African American woman presented with a 10-year history of photopsia, progressive decrease in visual acuity, metamorphopsia, and new nyctalopia. Both fundi showed evidence of periphlebitis, arterial attenuation, macular edema, and diffuse chorioretinal atrophy. RESULTS: Fluorescein angiography revealed diffuse vascular leakage, and indocyanine green showed evenly distributed and symmetrical hypofluorescent spots, which were difficult to appreciate on fundoscopy. Workup revealed a positive HLA-A29 and was negative for sarcoid, tuberculosis, and syphilis. CONCLUSION: Birdshot chorioretinopathy overwhelmingly affects non-Hispanic Caucasians, but there have been rare reported cases in other ethnicities including Hispanics and African Americans. This patient's ethnicity may have contributed to the 10-year delay in diagnosis. To our knowledge, this is the first documented HLA-A29 positive case of birdshot chorioretinopathy in an African American. HLA-A29 may be a useful supportive test in cases with classic clinical presentation in non-Caucasian patients to enable the correct diagnose in a timely manner.

Characterization of fetal antigen 1/delta-like 1 homologue expressing cells in the rat nigrostriatal system: effects of a unilateral 6-hydroxydopamine lesion.

Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.

Delivery of the p67 sporozoite antigen of Theileria parva by using recombinant Salmonella dublin: secretion of the product enhances specific antibody responses in cattle

The p67 sporozoite antigen of Theileria parva has been fused to the C-terminal secretion signal of Escherichia coli hemolysin and expressed in secreted form by attenuated Salmonella dublin aroA strain SL5631. The recombinant p67 antigen was detected in the supernatant of transformed bacterial cultures. Immunization trials in cattle revealed that SL5631 secreting the antigen provoked a 10-fold-higher antibody response to p67 than recombinant SL5631 expressing but not secreting p67. Immunized calves were challenged with a 80% lethal dose of T. parva sporozoites and monitored for the development of infection. Two of three calves immunized intramuscularly with the p67-secreting SL5631 strain were found to be protected, whereas only one of three animals immunized with the nonsecreting p67-expressing SL5631 strain was protected. This is the first demonstration that complete eukaryotic antigens fused to the C-terminal portion of E. coli hemolysin can be exported from attenuated Salmonella strains and that such exported antigens can protect cattle against subsequent parasite challenge.

CHARACTERIZATION OF THE COLONIZATION FACTOR ANTIGEN II PLASMID (CFA/II) FROM ENTEROTOXIGENIC ESCHERICHIA COLI

The etiological role of enterotoxigenic E. coli (ETEC) in diarrheal diseases of man and domestic animals is firmly established. Besides the production of enterotoxins (ST and LT), ETEC produces other important virulence factors; the colonization factor antigens (CFAs). CFAs mediate the attachment of ETEC to the epithelial cells of the small intestine, and this favors colonization by the bacteria and facilitates delivery of the enterotoxins to the intestinal cells.^ The production of enterotoxin and CFA is determined by plasmids and has been found to be restricted to a select number of E. coli serotypes.^ In this work, plasmid DNA analysis was performed in twenty-three CFA/II-producing enterotoxigenic Escherichia coli strains and their spontaneous CFA/II-negative derivatives. In some cases, strains lost the high molecular weight plasmid and also the ability to produce CFA/II, ST and LT. In other cases there was a deletion of the plasmid, which produced strains that were CFA/II('-), ST('-), LT('-) or CFA/II('-), ST('+), LT('+).^ The CFA/II plasmid from strain PB-176 (06:H16:CFA/II('+), ST('+), LT('+)) was transferred by transformation into E. coli K12 with concomitant transfer of the three characteristics: CFA/II, ST and LT.^ A physical map of the prototype CFA/II:ST:LT (pMEP60) plasmid was constructed by restriction endonuclease analysis and compared to plasmids from three other CFA/II-producing strains. A CFA/II-negative (but ST and LT positive) deletion derivative of pMEP60 (pMEP30) was also included in the map. The four CFA/II plasmids analyzed had a common region of approximately 30 kilobase pairs. The toxin genes were approximately 5 kbp apart and about 20 kbp from the common region. The information given by this physical map could be of great value when constructing a clone that will express the CFA/II genes but not the toxin genes. ^

STUDIES ON THE CLASS I GLYCOPROTEINS OF THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX (TRANSPLANTATION ANTIGEN, LYMPHOCYTE, CELL SURFACE MOLECULE)

A series of studies were undertaken to analyze and compare various aspects of murine class I glycoproteins. An initial area of investigation characterized the Qa-1 alloantigens using two-dimensional gel electrophoresis. Analysis of the products of the Qa-1('b), Qa-1('c) and Qa-1('d) alleles indicated that these were distinct molecules as determined by their lack of comigration upon comparative two-dimensional gel analysis. The importance of asparagine-linked glycosylation in the cell surface expression of class I molecules was also examined. These studies employed tunicamycin, an inhibitor of N-linked glycosylation. Tunicamycin treatment of activated T lymphocytes diminished the surface expression of Qa-1 to undetectable levels; the levels of other class I molecules exhibited little or no decrease. These results indicated that N-linked glycosylation has a differential importance in the cell surface expression of various class I molecules. The molecular weight diversity of class I molecules was also investigated. Molecular weight determination of both the fully glycosylated and unglycosylated forms of H-2 and Qa/Tla region encoded molecules established that there is a significant variation in the sizes of these forms of various class I molecules. The most significant difference ((TURN)9,000 daltons) exists between the unglycosylated forms of H-2K('b) and Qa-2, suggesting that the structural organization of these two molecules may be very different. A comparative two-dimensional gel analysis of various class I glycoproteins isolated from resting and activated T and B lymphocytes indicated that class I molecules expressed on activated T cells exhibited an isoelectrophoretic pattern that was distinct from the isoelectrophoretic pattern of class I molecules expessed on the other cell populations. This difference was attributed to a lower sialic acid content of the molecules expressed on activated T cells. Analysis of cell homogenates determined that activated T cells contained a higher level of endogenous neuraminidase activity than was detected in the other populations, suggesting that this may be the basis of the lower sialic acid content. The relationship of the Qa-4 and Qa-2 alloantigens was also examined. It was established that upon mitogen activation, the expression of Qa-4 was greatly decreased, whereas Qa-2 expression was not decreased. However, an anti-Qa-2 monoclonal antibody blocked the binding of an anti-Qa-4 monoclonal antibody to resting cells. These studies established that Qa-4 is a determinant restricted to resting cells, which is closely associated on the surface with the Qa-2 molecule. ^

DNA vaccination against the novel tumor antigen, MUC18, for the therapy of metastatic melanoma

Melanoma patients with metastases have a very low survival rate and limited treatment options. Therefore, the targeting of melanoma cells when they begin to invade and metastasize would be beneficial. A specific adhesion molecule that is upregulated at the vertical growth phase is the melanoma cell adhesion molecule (MCAM/MUC18). MUC18 is expressed in late primary and metastatic melanoma with little or no expression on normal melanocytes. MUC18 has been demonstrated to have a role in the progression and metastasis of human melanoma. We utilized the alphavirus-based DNA plasmid, SINCp, encoding full length human MUC18 for vaccination against B16F10 murine melanoma cells expressing human MUC18. The alphavirus-based DNA plasmid leads to the expression of large quantities of heterologous protein as well as danger signals due to dsRNA intermediates produced during viral replication. In a preventative primary tumor model and an experimental tumor model, mice vaccinated against human MUC18 had decreased tumor incidence and reduced lung metastases when challenged with B16F10 murine melanoma cells expressing human MUC18. In a therapeutic tumor model, vaccination against human MUC18 reduced the tumor burden in mice with pre-existing lung metastases but did not have a significant effect on therapeutic vaccination in a primary tumor model. We next cloned murine MUC18 into SINCp for use in determining the efficacy of vaccination against murine MUC18 in a syngeneic animal model. Mice were vaccinated and challenged in a primary tumor and experimental metastasis model. In both models, vaccination significantly reduced tumor incidence and lung metastases. Humoral and cell-mediated responses were then determined. Flow cytometry and immunohistochemistry showed that specific antibodies were developed from vaccination against both human and murine MUC18. IgG2a antibody isotype was also developed indicating a Th1 type response. ELISPOT results showed that mice vaccinated against human MUC18 created a specific T cell response to targets expressing human MUC18. Mice vaccinated against murine MUC18 raised specific effector cells against target cells expressing murine MUC18 in a cell killing assay. These results indicate that vaccination against MUC18 developed specific immune responses against MUC18 and were effective in controlling tumor growth in melanoma expressing MUC18. ^

Activation of dendritic cells from patients with epithelial ovarian cancer

Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing immune responses to tumor cells. Lin−HLA-DR + DC populations in peripheral blood mononuclear cells (PBMCs) and in ascites mononuclear leukocytes (MNLs) of patients with epithelial ovarian cancer (EOC) are phenotypically immature. Lin−HLA-DR + DCs from PBMCs of normal subjects and EOC patients and MNLs from ascites cells of patients were examined for specific cell surface markers or indicators of differentiation or activation. Separating Lin− HLA-DR+ DCs into subsets based on their HLA-DR intensity provided an additional method for identifying the two major lineages of DCs, myeloid and plasmacytoid. The activation potential of these DCs following exposure to the maturation agents CD40 ligand (CD40L) and lipopolysaccharide (LPS) was examined by measurement of IL-12 and IL-10 concentrations in DC culture supernatants in addition to their ability to stimulate allogeneic T cells. DCs from PBMCs of normal subjects and EOC patients and DCs isolated from ascites MNLs of EOC patients were separated into subsets based on CD11c and CD123 cell surface marker expression identifying the major DC types. These subsets were then compared with cells sorted on the basis of HLA-DR intensity. The in vivo behavior of DCs and DC subsets in peripheral blood and ascites following treatment of peritoneal carcinoma patients with the growth factor fins-like tyrosine kinase 3 ligand (Flt3L) was also examined. Increases in proportions and total numbers of DCs from peripheral blood and ascites were associated with increased secretion of IL-12 and IL-10 following in vitro activation of cultured DCs. There were differences between DCs from PBMCs and ascites and between DC subsets in expression of cell surface markers, cytokine profile, and the ability of Lin−HLA-DR + cells to stimulate proliferation of allogeneic T cells from EOC patients. These Lin−HLA-DR+ cells have certain functional properties that suggest that they could have the potential to facilitate an adaptive anti-tumor immune response. ^

Antigen-specific proteolytic antibodies

The antigen recognition site of antibodies is composed of residues contributed by the variable domains of the heavy and light chain subunits (VL and VH domains). VL domains can catalyze peptide bond hydrolysis independent of VH domains (Mei S et al. J Biol Chem. 1991 Aug 25;266(24):15571-4). VH domains can bind antigens noncovalently independent of V L domains (Ward et al. Nature. 1989 Oct 12;341(6242):544-6). This dissertation describe the specific hydrolysis of fusion proteins containing the hepatitis C virus coat protein E2 by recombinant hybrid Abs composed of the heavy chain of a high affinity anti-E2 IgG1 paired with light chains expressing promiscuous catalytic activity. The proteolytic activity was evident from electrophoresis assays using recombinant E2 substrates containing glutathione S-transferase (E2-GST) or FLAG peptide (E2-FLAG) tags. The proteolytic reaction proceeded more rapidly in the presence of the hybrid IgG1 compared to the unpaired light chain, consistent with accelerated peptide bond hydrolysis due to noncovalent VH domain-E2 recognition. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. Binding studies confirmed that the hybrid IgG retained detectable noncovalent E2 recognition capability, although at a level smaller than the wildtype anti-E2 IgG. Immunoblotting of E2-FLAG treated with the hybrid IgG suggested a scissile bond within E2 located ∼11 kD from the N terminus of the protein. E2-GST was hydrolyzed by the hybrid IgG at peptide bonds located in the GST tag. The differing cleavage pattern of E2-FLAG and E2-GST can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. This is the first proof-of principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit. ^