Natalie Fadle

Molecular definition of a novel human galectin which is immunogenic in patients with Hodgkin's disease.

Using autologous serum for the immunoscreening of a cDNA expression library derived from tissue involved by Hodgkins disease, a new 36-kDa protein with the characteristics of galectins (S-type lectins) was detected. Sequence analysis of the cDNA clone HOM-HD-21 revealed two homologous motifs known as lectin domains with galactoside binding capacity. The two domains are linked by a stretch of about 30 amino acid residues and share a sequence homology of 39%. While the N-terminal lectin domain shows merely moderate homologies with known galectins, the C-terminal lectin domain is highly homologous to rat galectin-5 with an amino acid sequence identity of 70%. We ruled out mutations of the tumor-derived transcript by sequence comparison with the respective cDNA cloned from normal peripheral blood leukocytes. Recombinant protein expressed in Chinese hamster ovary cells was purified from lysates by lactose and galactose affinity chromatography, proving the galactoside binding capacity of this new galectin. Northern blot analysis revealed an expression spectrum restricted to peripheral blood leukocytes and lymphatic tissues. In accordance with the nomenclature of known galectins, we suggest to designate this novel galactoside binding protein galectin-9.

EB/RP gene family encodes tubulin binding proteins

Mutations in the adenomatous polyposis coli (APC) gene are linked to the dysplastic transformation of colorectal polyps and represent an early step in the development of colorectal tumors. Ninety‐four percent of all mutations result in the expression of a truncated APC protein lacking the C‐terminal region. The C‐terminal region of the APC protein may have a tumor suppressor function as its absence appears to be linked to the development of dysplastic lesions. Recently, we discovered and characterized a protein called RP1 which binds specifically to the C‐terminal region of the APC protein. We show now that RP1 and the other known members of the EB/RP family (EB1 and RP3) also bind directly to tubulin, both in vitro and in vivo. Immunohistochemical analyses reveal a distinct staining pattern during interphase as well as an association of RP1/EB1 with mitotic microtubule structures. The previously described puncta of the APC protein at the leading edge of membrane protrusions contact microtubule fibers that contain RP1 or EB1. Int. J. Cancer 81:275–284, 1999.

Association of a dominantly inherited hyperphosphorylated paraprotein target with sporadic and familial multiple myeloma and monoclonal gammopathy of undetermined significance: a case–control study

BACKGROUND Chronic antigenic stimulation might have a role in the pathogenesis of monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma. The aim of this study was to search for factors underlying the autoimmunogenicity of paratarg-7, a frequent antigenic target of paraproteins in MGUS and multiple myeloma. METHODS Between January, 2005, and February, 2009, serum and peripheral blood cells were obtained from consecutive patients with MGUS or multiple myeloma and healthy blood donors, and paratarg-7 was analysed by DNA sequencing, SDS-PAGE, isoelectric focusing, and western blotting. FINDINGS Mutations or polymorphisms of paratarg-7 were not noted, but hyperphosphorylation was detected in 35 (13.9%) of 252 patients with MGUS or multiple myeloma, all of whom had an anti-paratarg-7-specific paraprotein. Analysis of eight families showed that hyperphosphorylated paratarg-7 is inherited in a dominant fashion, and that carriers of hyperphosphorylated paratarg-7 have an increased risk of developing MGUS and multiple myeloma (odds ratio [OR] 7.9, 95% CI 2.8-22.6; p=0.0001). INTERPRETATION Familial MGUS and multiple myeloma were associated with a dominant inheritance of hyperphosphorylated paratarg-7, enabling family members at increased risk for MGUS or multiple myeloma to be identified. That only patients with MGUS or multiple myeloma who are carriers of hyperphosphorylated paratarg-7 had a paratarg-7-specific paraprotein suggests that the hyperphosphorylation of paratarg-7 induces auto-immunity and is involved in the pathogenesis of MGUS and multiple myeloma; for example, by chronic antigenic stimulation. FUNDING Förderverein Krebsforschung Saar-Pfalz-Mosel e.V. (eingetragener Verein: officially registered charity) and HOMFOR (the research programme of the Saarland University Faculty of Medicine).

Progranulin antibodies in autoimmune diseases

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasus arteritis (4/13), classical panarteritis nodosa (4/10), Behcets disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

Targeted Bioactivity of Membrane-Anchored TNF by an Antibody-Derived TNF Fusion Protein

We describe the generation and characterization of a fusion protein consisting of a humanized anti-fibroblast-activating protein (anti-FAP) Ab and human TNF replacing the IgG1 CH2/CH3 Fc domain. The construct was generated by recombinant DNA technology and preserved its IgG1-derived dimeric structure with the TNF molecule linked as a dimer. Expression in CHO cells was optimized in serum-free medium under GMP conditions to achieve production levels up to 15 mg/liter. Recognition of the FAP Ag by the construct was as good as that by the parental anti-FAP Ab. TNF signaling was induce able via both TNF receptor types. When acting in solution, the Ab-linked TNF dimer exhibited a 10- to 20-fold lower activity compared with recombinant trimeric TNF. However, after binding to FAP-expressing cells, immobilized anti-FAP-TNF dimer was equivalent to membrane-anchored TNF with regard to bioactivity. Amplification of TNF-related pathways by mimicking the membrane-integrated TNF signaling was detectable in various systems, such as apoptosis induction or tissue factor production. The difference in TNF receptor type 1 and 2 signaling by the anti-FAP-TNF construct correlated well with its Ag-bound or -soluble status. Translating the approach into a xenograft animal model (BALB/c nu/nu mice), we demonstrated low toxicity with measurable antitumor efficacy for the TNF fusion protein after i.v. application. Immunohistochemical analysis of tumor sections showed restricted TNF-mediated macrophage recruitment to the targeted tissue in a time- and dose-dependent manner. These data warrant transfer of the anti-FAP-TNF immunocytokine into clinical trials for the treatment of FAP-positive tumors.

A frequent target of paraproteins in the sera of patients with multiple myeloma and MGUS

Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins might play a role in the pathogenesis of these neoplasms. We screened a human fetal brain‐derived macroarray with the IgA or IgG containing sera of 192 consecutive MGUS and MM patients. Twenty‐nine of 192 (15.1%) paraproteins reacted with paratarg‐7, a protein of unknown function which is expressed in all human tissues. Paratarg‐7 reactivity was similarly frequent among IgA and IgG paraproteins, but all paratarg‐7 reactive IgG paraproteins belonged to the IgG3 subtype with 24/57 IgG3 (42.1%) paraproteins displaying this specificity. Sequence analysis of paratarg‐7 derived from patients having a paraprotein with specificity for paratarg‐7 revealed no differences to paratarg‐7 derived from patients with paraproteins of other specificities or healthy controls, excluding mutations or polymorphisms as a reason for its autoimmunogenicity. Similarly, Western‐blot analysis showed identical bands for paratarg‐7 derived from patients and controls. The above‐random frequency of paratarg‐7 as a paraprotein target suggests that paratarg‐7 might be involved in the development of the respective clonal proliferations. The identification of paratarg‐7 as an antigenic target enables the more detailed analysis of tumor–host interactions in these patients and their role in the pathogenesis of MM and MGUS.

Hyperphosphorylated paratarg-7: a new molecularly defined risk factor for monoclonal gammopathy of undetermined significance of the IgM type and Waldenström macroglobulinemia

We recently described paratarg-7 (P-7), a protein of unknown function, as the target of 15% of immunoglobulin A (IgA) and IgG paraproteins in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma. To determine the frequency of P-7 as a paraprotein target in IgM-MGUS and Waldenström macroglobulinemia (WM), sera from patients with IgM-MGUS/WM were tested for reactivity with recombinant P-7 by enzyme-linked immunoabsorbent assay. The specificity of the paraprotein-mediated reaction was shown by absorption studies and cloning of the respective B-cell receptor. The paraproteins of 18 (9 WM and 9 IgM-MGUS) of 161 patients (11%) reacted with P-7. Isoelectric focusing and phosphatase treatment showed that P-7 was hyperphosphorylated (pP-7) in all patients with an anti-P-7-specific IgM paraprotein tested. Because only 4 of 200 healthy controls (2%) were carriers of pP-7, pP-7 carrier state is associated with a significantly increased risk (odds ratio = 6.2; P = .001) for developing IgM-MGUS/MW. Family analyses showed that the pP-7 carrier state is inherited as a dominant trait. After IgA/IgG-MGUS and multiple myeloma, IgM-MGUS/WM is the second neoplasia associated with pP-7 carrier state. The dominant inheritance of pP-7 explains cases of familial IgM-MGUS/WM and enables the identification of family members at increased risk.

Progranulin antibodies entertain a proinflammatory environment in a subgroup of patients with psoriatic arthritis

IntroductionPsoriatic arthritis (PsA) is a distinctive inflammatory arthritis which may typically develop in a subgroup of individuals suffering from psoriasis. We recently described progranulin autoantibodies (PGRN-Abs) in the sera of patients with different autoimmune diseases including seronegative polyarthritis. In the present study we investigated the occurrence of PGRN-Abs in PsA.MethodsPGRN-Abs were determined in 260 patients with PsA, 100 patients with psoriasis without arthritic manifestations (PsC) and 97 healthy controls using a recently described ELISA. PGRN plasma levels were determined from subgroups by a commercially available ELISA-kit. Possible functional effects of PGRN-antibodies were analysed in vitro by tumour necrosis factor (TNF)-α mediated cytotoxicity assays using WEHI-S and HT1080 cells.ResultsPGRN-Abs were detected with relevant titres in 50/260 (19.23%) patients with PsA, but in 0/100 patients with psoriasis without arthritic manifestations (P = 0.0001). All PGRN-Abs belonged to immunoglobulin G (IgG). PGRN-Abs were significantly more frequent in PsA patients with enthesitis or dactylitis. PGRN-Abs were also more frequent in PsA patients receiving treatment with TNF-α-blockers than in patients treated without TNF-α-blockers (20.8% versus 17.4%; P = 0.016). PGRN plasma levels were significantly lower in PGRN-Ab-positive patients with PsA than in healthy controls and patients with psoriasis without arthritic manifestations (P < 0.001), indicating a neutralizing effect of PGRN-Abs. Moreover cytotoxicity assays comparing PGRN-antibody positive with negative sera from matched patients with PsA, clearly showed a proinflammatory effect of PGRN antibodies.ConclusionNeutralizing PGRN-Abs occur with relevant titres in a subgroup of patients with PsA, but not in patients without arthritic manifestations (PsC). PGRN-Ab-positive patients had more frequent enthesitis or dactylitis. TNF-α-induced cytotoxicity assays demonstrated that the protective effects of progranulin were inhibited by serum containing PGRN-Abs. This suggests that PGRN-Ab might not only be useful as a diagnostic and prognostic marker, but may provide a proinflammatory environment in a subgroup of patients with PsA.

Autoantigenic targets of B-cell receptors derived from chronic lymphocytic leukemias bind to and induce proliferation of leukemic cells.

Antigenic targets of the B-cell receptor (BCR) derived from malignant cells in chronic lymphocytic leukemia (CLL) might play a role in the pathogenesis of this neoplasm. We screened human tissue-derived protein macroarrays with antigen-binding fragments derived from 47 consecutive cases of CLL. An autoantigenic target was identified for 12/47 (25.5%) of the cases, with 3 autoantigens being the target of the BCRs from 2 patients each. Recombinantly expressed autoantigens bound specifically to the CLL cells from which the BCR used for the identification of the respective autoantigen was derived. Moreover, binding of the autoantigen to the respective leukemic cells induced a specific activation and proliferation of these cells. In conclusion, autoantigens are frequent targets of CLL-BCRs. Their specific binding to and induction of proliferation in the respective leukemic cells provide the most convincing evidence to date for the long-time hypothesized role of autoantigens in the pathogenesis of CLL.

Mitofilin and titin as target antigens in melanoma-associated retinopathy.

Melanoma‐associated retinopathy (MAR) is a rare paraneoplastic syndrome in patients with melanoma. Since the onset of MAR symptoms is often associated with tumor progression or recrudescence of metastases, MAR‐related symptoms are prognostic relevant. The pathomechanism underlying MAR is supposed to result from antibody production against yet unknown melanoma‐associated antigens that are also expressed in retinal tissue, leading to the destruction of retinal cells and resulting in defective signal transduction. Only a 35 kDa protein in Müller glial cells, a 22 kDa neuronal antigen and retinal transducin have been identified as MAR‐associated antigens to date. To identify additional antigens potentially involved in the pathogenesis of MAR, we screened a retina cDNA phage library for reactivity with antibodies in the sera from 9 patients with MAR or subclinical MAR using the serological analysis of recombinantly expressed clones (SEREX) approach. Six sera from melanoma patients without evidence of MAR and 10 sera from healthy donors served as controls. Mitofilin and titin were identified as antigens against which antibodies were found exclusively in sera of MAR patients, but not in the sera of MM patients without MAR or healthy donors. This is the first study to demonstrate that titin is highly expressed from retinal tissue and melanoma. The fact that none of the MAR‐associated antigens detected to date by their capacity to elicit a humoral immune response is located on the cell surface questions a major pathogenetic role of the respective antibodies and suggests that cellular, rather than humoral mechanisms are operative in the primary immune attack against the retina in MAR.