43-kD Protein Research Articles

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

Diagnostic Features of Pemphigus vulgaris in Patients with Bullous Pemphigoid

Background: The simultaneous presence of features of pemphigus vulgaris (PV) in patients with bullous pemphigoid (BP) has previously been reported in the literature. Objective: The purpose of this retrospective study is to present 13 patients with an initial diagnosis of BP, who subsequently demonstrated coexistent serological features of both BP and PV. Methods: The following information on each patient was documented, at the time of initial diagnosis: clinical profile on presentation, histology, direct immunofluorescence, indirect immunofluorescence (IIF) using monkey esophagus as substrate, salt-split skin (SSS) and an immunoblot assay. Since all 13 patients failed to respond to conventional systemic therapy, intravenous immunoglobulin (IVIg) was used as an alternative treatment modality. Prior to initiating IVIg therapy, in all 13 patients, serological studies were performed. In addition to IIF using monkey esophagus, an immunoblot assay and SSS, an enzyme-linked immunosorbent assay (ELISA) was performed to detect antibodies to desmogleins. These different assays were done to identify pathological autoantibodies typical of BP and PV. A control group of 25 healthy normal individuals, 37 patients with BP, 17 patients with PV and 12 patients with pemphigus foliaceus were used for comparison of serological studies. Results: At the time of initial presentation, histological and immunopathological studies confirmed the diagnosis of BP in all 13 patients. Prior to the initiation of IVIg therapy, results of IIF using monkey esophagus as substrate demonstrated high levels of anti-intercellular cement substance (anti-ICS) or antikeratinocyte cell surface antibody. Sera of all 13 patients on SSS bound to the epidermal side of the split. In an immunoblot, using bovine gingival lysate as substrate, sera of 6 patients bound to both a 230-kD (BP Ag1) and 180-kD protein (BP Ag2), while 7 sera bound to only a 230-kD protein. All 13 patients had high levels of antibodies to desmoglein 3 on ELISA. In a pilot experiment, the anti-ICS antibody in sera from 6 random patients was found to be predominantly of the IgG4 subclass. Use of IVIg resulted in an effective clinical response and the maintenance of a prolonged clinical remission. Conclusion: In patients with BP, who are nonresponsive to conventional therapy, the presence of two autoimmune diseases or a dual diagnosis should be considered.

Procollagen-Like Protein as a Molecular Target in the Treatment of Primary Brain Tumor.

METHODS. cDNA library was obtained in one glioma cell line which highly expressed this protein and screening was performed using VDR monoclonal antibody. This partial cDNA was then subcloned into pcDNA3.1 and sequencing was performed. This partial cDNA sequence also was used as a probe to identify mRNA, which encodes for this putative gene. To characterize this 220-Kd protein, we have used five cell lines as follows: Glioma 1, U118, SW1783, which possess variable amount of 220-Kd protein, and U-373 and T-98G which lack this 220-Kd protein.

Inhibition of Platelet Aggregation by Rat Trophoblasts

Objective: To determine the effect of rat trophoblast cell suspensions on adenosine diphosphate (ADP)-induced platelet aggregation. Materials and Methods: The trophoblasts were isolated from ectoplacental cones (a pre-placental tissue very rich in trophoblasts) developed in rat embryos on day 12 of pregnancy (normal period of gestation: 22–23 days). Platelet-rich plasma (PRP) was obtained from adult male rats. The trophoblasts were preincubated (37°C, 30 min), suspended in the medium, and then re-incubated with PRP for 3–5 min. Results: 5 × 10⁴ and 7 × 10⁴ trophoblasts inhibited ADP-induced platelet aggregation by 10 and 18%, respectively. When the trophoblast cell concentration was increased to 1– 2 × 10⁵ cells, a proaggregatory phenomenon was observed, even in the absence of ADP. However, there was no inhibition of aggregation or promotion of aggregation when fixed trophoblasts or live endometrial stromal cells were incubated with PRP. Conclusion: The results indicate that the aggregation inhibition response was cell specific and concentration dependent. A 68-kD protein was detected in the medium when it was conditioned with 5–7 × 10⁴ but not with 1–2 × 10⁵ trophoblasts. However, the inhibitory or stimulatory effect does not seem to be dependent on the presence of this protein.

Insulin-induced glycosylphosphatidylinositol (GPI) binding to red cell membrane proteins

In this work GPI binding to membrane proteins from erythrocytes of insulinoma patients for whom prolonged hyperinsulinism and hypoglycemia were characteristic, as well as from normal erythrocytes incubated with supraphysiological concentrations of insulin were analyzed. In the RBCs from insulinoma patients, covalent GPI binding to red cell membrane proteins in the spectrin/ankyrin region, band 4.1 and two proteins of molecular mass of 115 and 110 kD was demonstrated. In erythrocytes incubated with insulin label was associated with band 4.1 and two proteins of molecular mass of 115 and 110 kD. Extraction studies showed that the 100-kD proteins are unrelated to band 3 since they were found in Triton- prepared cytoskeleton. To our knowledge this is the first demonstration of such a modification of red cell skeletal proteins, and the first demonstration of post-translation GPI binding to red cell skeletal proteins in response to insulin. A mechanism proposed for GPI binding to red cell skeletal proteins as well as the relevance of these results for physiological disorders that are characterized by hyperinsulinism are briefly discussed.

The Genome Organization of Pea Stem Necrosis Virus and Its Assignment to the Genus Carmovirus

The complete sequence of the single-stranded, positive-sense RNA genome of pea stem necrosis virus (PSNV) has been determined. The 4,048-nucleotide genome contains five open reading frames (ORFs). The 5′-proximal ORF encodes a 25-kD protein (p25). If the amber termination codon is read through, the ORF produces a read-through protein of 84 kD (p84). Two small, centrally located ORFs encoded a 7-kD protein (p7) and a 6-kD protein (p6), respectively. The 38-proximal ORF encodes a 38-kD (p38) capsid protein. Comparison of the genome organization with that of other viruses justifies the assignment of PSNV to the genus Carmovirus.

The antiviral protein human lactoferrin is distributed in the body to cytomegalovirus (CMV) infection-prone cells and tissues.

Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving rats. Furthermore, the systemic availability of lactoferrin after intraperitoneal dosing was determined. After intravenous injection, human lactoferrin (hLF) was rapidly cleared from the plasma, but higher doses resulted in prolonged plasma levels. Immunohistochemical analysis revealed a pronounced distribution of hLF to endothelial cells in the liver whereas diffuse staining in hepatocytes indicated the presence of considerable amounts in this large cell population. This endothelial association, which also was found in other organ/tissues, including blood vessels. was confirmed by in vitro cell-binding studies. In addition, leukocytes in plasma that were infiltrated in various organs showed binding of hLF. A small fraction of hLF was transported into the lymphatic system. Western blot analysis revealed that hLF, present in the various organs. mainly consisted of an 80-kD protein. After intraperitoneal administration, small amounts of 80-kD hLF distributed to the general circulation. The bioavailability was 0.6% but increased to 3.6% after multiple administrations. The affinity of hLF for endothelial cells and leukocytes, and its penetration into the lymphatic system, indicates that this protein reaches target cells and body compartments that are crucial for CMV and HIV replication. The ability to reach the blood compartment after intraperitoneal dosing offers opportunities for parenteral administration of the protein in future studies on its antiviral effects in vivo.

Expression, characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen, IA-2, in Sf-9 cells.

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.

Antibodies against a novel nucleolar and cytoplasmic antigen (p105-p42) present in the sera of patients with a subset of rheumatoid arthritis (RA) with signs of scleroderma.

We identified three patients (two of them relatives) with RA and signs of scleroderma whose sera contained a high titre of IgG class antibodies against the nucleoli and the nucleoplasm of cells of different mammalian origins. Sera from these patients uniformly immunoprecipitated four polypeptides, from a 35S-methionine-labelled HeLa cell extract, whose mol. wts were 120, 105, 95 and 42 kD. Of these, the 95-kD protein was highly phosphorylated. By immunoblotting, these sera reacted with 105-, 95- and 42-kD proteins and affinity-purified antibodies from these, demonstrating that 105- and 95-kD proteins shared cross-reactive epitopes. Moreover, affinity-purified antibodies from each of these proteins immunoprecipitated the whole complex. Localization studies using immunoelectron microscopy and in vivo actinomycin-D-treated cells demonstrated that the 105-, 95- and 42-kD proteins were present in the granular component of the nucleolus and the nucleoplasm. In addition, the 105- and 95-kD were present in free polyribosomes as well as ribosomes attached to endoplasmic reticulum. Pulse/chase experiments strongly suggested that the complex was accomplished shortly after a 10-min pulse. It was preferentially present in the nucleus after a 2 h chase and in both nucleus and cytoplasm after a 5 h chase. We conclude that a protein complex with a main nucleolar distribution is a new autoantigen (p105-p42) recognized by autoantibodies present in the serum of a subgroup of patients with RA and scleroderma signs. These antibodies could be useful as diagnostic markers and as tools for further studies involving the biology of the nucleolus.

IgG subclass distribution of antibody responses to protein and polysaccharide mycobacterial antigens in leprosy and tuberculosis patients.

Immunoenzymatic assays were developed for the measurement of antibodies against mycobacterial lipoarabinomannan (LAM), a cell-free proteic extract (CFX) of Mycobacterium leprae, and the 38-kD protein antigen of M. tuberculosis. Sera from 108 leprosy patients, belonging to all clinical-immunological forms of the spectrum, and 81 patients with localized or disseminated tuberculosis (TB) were tested for antibodies of the four IgG subclasses. Standard calibration curves were used to allow comparisons between results of different isotypes and specificities. Mean concentrations of total IgG antibodies were higher in the overall leprosy population than in TB patients. In leprosy, levels of anti-CFX increased from tuberculoid toward lepromatous forms, with a clear switch from IgG1 to IgG2 subclass predominance. A similar IgG1 to IgG2 conversion was observed in anti-LAM antibodies, although total levels of anti-LAM were similar in patients with tuberculoid and lepromatous forms. In TB, antibodies against polysaccharide and protein antigens were both predominantly of IgG1 subclass, whatever the patient's clinical status, although lower in disseminated forms, probably due to concomitant HIV infection. A hypergammaglobulinaemia was also found in most leprosy and TB patients. In TB this was due to increased IgG1 and IgG3, especially in HIV co-infected patients. Based on the current knowledge of the influence of T cell-secreted cytokines on human immunoglobulin isotype expression, these results do not fit with a putative role of Th1 (such as found in TB and tuberculoid leprosy (TT)) and Th2 (such as found in leprosy lepromatous (LL) leprosy) environment in the isotypy of antibody responses in mycobacterial infections. Nor do variations of isotypy according to pathological conditions seem to be related to the biochemical nature of antigens, since antibodies to LAM and protein antigens had comparable evolutions of their subclass distribution. Other factors are to be investigated in order to understand better the significance and possible roles of antibodies in mycobacterial diseases.

Ro/SS-A- and La/SS-B-reactive B lymphocytes in peripheral blood of patients with Sjögren's syndrome.

The aim of this study was to investigate the production of anti-Ro/SS-A and anti-La/SS-B antibodies in peripheral blood (PB) of patients with Sjögren's syndrome (SS). The ELISPOT method was performed to quantify the frequency of PB lymphocytes spontaneously secreting anti-Ro/SS-A and/or anti-La/SS-B antibodies. The total number of IgG-, IgA- and IgM-producing cells was also quantified. The recombinant Ro 52-kD, Ro 60-kD and La 48-kD proteins were used as target antigens. Three of 18 SS patients had PB lymphocytes secreting IgG antibodies against the recombinant Ro 52-kD protein. The same three patients had high serum titres of anti-Ro 52-kD antibodies. In addition, these patients were classified as having severe disease, and all three had focus scores of >/= 8 in biopsies of the labial salivary glands (LSG). The correlation between the number of PB cells producing IgG antibodies against the recombinant Ro 52-kD protein and the focus score was significant (P < 0.01). The results indicate that only SS patients with severe disease and high degree of local inflammation in LSG have B cells producing anti-Ro/SS-A antibodies in PB. Thus, most of the spontaneous autoantibody production must take place in other body compartments, e.g. in exocrine glands and probably also in the lymphoid organs and/or other mucosal sites.

Analysis of human C4A and C4B binding to an immune complex in serum.

Previous studies using isolated complement proteins have shown that more C4A than C4B binds to certain types of immune complexes. However, the in vivo binding of the C4 isoforms to an immune complex has not been investigated in detail and may differ from events when measured with the isolated proteins. We report here the binding of C4A and C4B to an immune complex of bovine serum albumin (BSA) anti-BSA as it occurs in serum. We found that when using the isolated C4 proteins more C4A than C4B bound to the complex, but in serum similar amounts of C4A and C4B were found to bind. Furthermore, these results were not explainable by a difference in activity between isoforms. In an attempt to explain these results a number of unexpected observations were noted. First C4A, but not C4B, bound specifically to a yet unidentified 38-kD serum protein. Second, when both covalent and non-covalent binding was assessed, we found that as serum concentration increased there followed a concomitant decrease in covalent binding and C4B was more affected than C4A. The potential biological significance of these findings is discussed.

Association of neutropenia in systemic lupus erythematosus (SLE) with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen.

SLE is associated with the production of autoantibodies to self-constituents. In particular, certain ribonucleoprotein particles are targeted. Despite the multitude of autoantibodies produced and the remarkable concentrations of these antibodies in the sera of SLE patients, there have been little data that the autoantibodies found in SLE are involved in the pathogenesis of disease or its manifestations. The present work demonstrates that anti-Ro (or SSA) is associated with granulocytopenia, binds the surface of granulocytes and fixes complement to this membrane surface. Binding is a property of anti-Ro Fab fragments and can be inhibited by 60-kD Ro. However, the antigen bound on the surface of granulocytes is a 64 000 mol. wt protein that is a novel autoantigen in SLE. As suggested by inhibition studies, sequence identity between 60-kD Ro and eight tandem repeats in the 64-kD antigen may be responsible for the observed serologic cross-reactivity. These data imply that anti-Ro antibodies that also bind the 64-kD protein mediate neutropenia in patients with SLE.

Correction of cross-linker sensitivity of Fanconi anemia group F cells by CD33-mediated protein transfer

AbstractStudies have previously described the feasibility of receptor-mediated protein transfer in a cell culture model of Fanconi anemia (FA) group C. This study explores the versatility of this approach by using an antibody single-chain fusion protein to correct the phenotypic defect in FA group F cells. A 68.5-kd chimeric protein (His-M195FANCF) was expressed, consisting of a His tag, a single-chain antibody to the myeloid antigen CD33, and the FANCF protein, as well as a 43-kd His-FANCF fusion protein lacking the antibody motif, inEscherichia coli. The nickel-agarose–purified His-M195FANCF protein bound specifically to the surface of HeLa cells transfected with CD33 and internalized through vesicular structures. The fusion protein, but not CD33, sorted to the nucleus, consistent with the known nuclear localization of FANCF. No similar binding or internalization was observed with His-FANCF. Pretreatment of the transfected cells with chloroquine abolished nuclear accumulation, but there was little change with brefeldin A, indicating a minimal if any role for the Golgi apparatus in mediating transport from endosomes to the cytosol and the nucleus. The intracellular half-life of His-M195FANCF was approximately 160 minutes. Treatment of CD33-transfected FA group F lymphoblastoid cells with 0.1 mg/mL His-M195FANCF conferred resistance to mitomycin C. No similar protection was noted in CD33− parental cells or CD33+ FA cells belonging to groups A and C. These results demonstrate that antibody-directed, receptor-mediated protein transfer is a versatile method for the delivery of biologically active proteins into hematopoietic cells.

Ubiquitin Recognition and Chaperoning

The yeast transcription factor SPT23 is a homolog of the mammalian transcription factor nuclear factor-κB (NF-κB). In its immature form, SPT23 is a 120-kD protein tethered to the membrane of the endoplasmic reticulum (ER). The processing of p120 seems to involve the following: (i) ubiquitination and proteolytic cleavage to a mature 90-kD (p90) form, and (ii) the function of a protein complex consisting of CDC4--a protein involved in the post-mitotic fusion of Golgi membranes--and Udf1 and Npl4, which are thought to be involved in ubiquitin-dependent protein degradation and nuclear transport, respectively. However, the mechanism of SPT23 activation is largely unresolved. Rape et al. have found that p120 SPT23 must homodimerize at the ER membrane, as the first step in maturation. Yeast two-hybrid assays showed that the minimal region that conferred homodimerization was the immunoglobulin (Ig)-like-plexins-transcription factors (IPT) domain, and that IPT domains could associate with each other, suggesting that an IPT-IPT interaction was necessary and sufficient for SPT23 homodimerization. One of the SPT23 monomers became ubiquitinated by the ubiquitin ligase RSP5. The proteasome proteolytically cleaved the stalk that tethers SPT23 to the ER membrane. However, the cleaved SPT23, now p90, remained stably associated with the unprocessed monomer, as demonstrated by washing with a gradient of low-to-high salt concentrations. The removal of mature p90 from the immature monomer required the ATP-dependent activity of CDC48, which could discriminate the ubiquitinated p90 from p120. Additionally, the translocation of p90 to the nucleus was also CDC48-dependent, suggesting that CDC48 may act as a ubiquitin-selective chaperone protein. M. Rape, T. Hoppe, I. Gorr, M. Kalocay, H. Richly, S. Jentsch, Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48 UFD1/NPL4 , a ubiquitin-selective chaperone. Cell 107 , 667-677 (2001). [Online Journal]

A Slow Maturation of a Cysteine Protease with a Granulin Domain in the Vacuoles of Senescing Arabidopsis Leaves

Arabidopsis RD21 is a cysteine protease of the papain family. Unlike other members of the papain family, RD21 has a C-terminal extension sequence composed of two domains, a 2-kD proline-rich domain and a 10-kD domain homologous to animal epithelin/granulin family proteins. The RD21 protein was accumulated as 38- and 33-kD proteins in Arabidopsis leaves. An immunoblot showed that the 38-kD protein had the granulin domain, whereas the 33-kD protein did not. A pulse-chase experiment with Bright-Yellow 2 transformant cells expressing RD21 showed that RD21 was synthesized as a 57-kD precursor and was then slowly processed to make the 33-kD mature protein via the 38-kD intermediate. After a 12-h chase, the 38-kD intermediate was still detected in the cells. These results indicate that the N-terminal propeptide was first removed from the 57-kD precursor, and the C-terminal granulin domain was then slowly removed to yield the 33-kD mature protein. Subcellular fractionation of the Bright-Yellow 2 transformant showed that the intermediate and mature forms of RD21 were localized in the vacuoles. Under the acidic conditions of the vacuolar interior, the intermediate was found to be easily aggregated. The intermediate and the mature protein were accumulated in association with leaf senescence. Taken together, these results indicate that the intermediate of RD21 was accumulated in the vacuoles as an aggregate, and then slowly matured to make a soluble protease by removing the granulin domain during leaf senescence.

PH-induced folding of an apoptotic coiled coil.

Par-4 is a 38-kD protein pivotal to the apoptotic pathways of various cell types, most notably prostate cells and neurons, where it has been linked to prostate cancer and various neurodegenerative disorders including Alzheimer's and Huntington's diseases and HIV encephalitis. The C-terminal region of Par-4 is responsible for homodimerization and the ability of Par-4 to interact with proposed effector molecules. In this study, we show that the C-terminal 47 residues of Par-4 are natively unfolded at physiological pH and temperature. Evidence is rapidly accumulating that natively unfolded proteins play an important role in various cellular functions and signaling pathways, and that folding can often be induced on complexation with effector molecules or alteration of environment. Here we use primarily CD studies to show that changes in the environment, particularly pH and temperature, can induce the Par-4 C terminus to form a self-associated coiled coil.

Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins.

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and α-actinin and colocalizes with vinculin/metavinculin and α-actinin at microfilament attachment sites, such as cell–cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

Cytokeratin 7 and 20 and thyroid transcription factor 1 can help distinguish pulmonary from gastrointestinal carcinoid and pancreatic endocrine tumors

Expression of cytokeratin (CK) 7 and 20 is commonly used to help distinguish adenocarcinomas from different sites. Thyroid transcription factor 1 (TTF-1) is a 38-kd protein, located primarily in the nucleus of type 2 pneumocytes and clara cells. TTF-1 has been shown to be present in a variety of lung and thyroid tumors and in pulmonary small-cell carcinomas. Carcinoid tumors from the lung and the gastrointestinal (GI) tract are histologically similar and thus are difficult to differentiate from each other based on histologic criteria. Pancreatic endocrine tumors (PET) have a similar histologic appearance to these other tumors. The purpose of this study was to determine the efficacy of differentiating these 3 groups of tumors by their expression of CK7, CK20, and TTF-1. Routinely processed paraffin-embedded tissue sections from 62 carcinoid tumors (lung, 16; gastrointestinal [GI] tract, 46) and 12 PETs were immunohistochemically stained for CK7, CK20, and TTF-1. The degree of expression in each tumor was graded as 1+ (1% to 10% of cells positive), 2+ (11% to 25%), 3+ (26% to 50%), and 4+ (>50%). The data were compared between tumor types and between carcinoid tumors from the various locations in the GI tract (stomach, 8; small intestine, 19; large intestine, 17; appendix, 2). CK7 was expressed in 10 (63%) of 16 pulmonary carcinoid tumors and only 5 (11%) of 46 GI carcinoid tumors (P <.001). Pancreatic endocrine tumors showed CK7 positivity in 6 (50%) of 12 cases, which was similar to the findings in lung carcinoids and significantly higher than in GI carcinoids (P <.01). CK20 was expressed in 0 (0%) of 16 pulmonary carcinoid tumors, in contrast to 24% and 33% of GI carcinoid tumors (P <.05) and PETs (P <.05), respectively. TTF-1 expression was highly specific for pulmonary carcinoid tumors. This peptide was present in 11 (69%) of 16 pulmonary carcinoid tumors and in only 1 (2%) of 46 and 0 (0%) of 12 GI carcinoid tumors (P <.001) and PETs (P <.001), respectively. A CK7+/CK20−/TTF-1+ immunopanel result was moderately sensitive (sensitivity, 50%), and highly specific (specificity, 100%), for a diagnosis of pulmonary carcinoid tumor. CK7, CK20, and TTF-1 did not differ significantly between carcinoid tumors located in different sites of the GI tract. However, a trend was observed toward a lower prevalence of CK20 positivity in gastric tumors (P =.06) than in GI carcinoid tumors from the small intestine, colon, or appendix. Expression of CK7 and CK20, and particularly TTF-1, may be useful in distinguishing pulmonary from GI carcinoid tumors and PETs, especially when evaluated as a panel of markers. TTF-1 is highly specific for pulmonary carcinoid tumors. HUM PATHOL 32:1087-1093. Copyright © 2001 by W.B. Saunders Company

Immunoglobulin gene usage and immunohistochemical characteristics of human monoclonal antibodies to the mitochondrial autoantigens of primary biliary cirrhosis induced in the XenoMouse.

The immunodominant antimitochondrial antibody (AMA) response in primary biliary cirrhosis (PBC) is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). The nature of the clonal selection process is unclear, and to address this issue, we took advantage of a transgenic technology, XenoMouse, that contains 80% of the human immunoglobulin (Ig) variable gene repertoire and can produce high-affinity human antibodies to virtually any immunogen without evidence of clonal bias. We immunized mice with PDC-E2 to obtain 13 HmAbs, including 4 IgG(2) and 9 IgM isotypes. Immunoglobulin gene analysis was unique and demonstrated a clonal bias; the immunoglobulin gene usage was considerably different from other antibody responses analyzed in XenoMouse systems. Four of the 13 mAbs recognized the inner lipoyl domain of PDC-E2, 2 of 13 recognized the entire PDC-E2 molecule, 4 of 13 recognized PDC-E2 and OGDC-E2, 1 of 13 recognized OGDC only, 1 recognized BCOADC-E2 only, and 1 recognized an unidentified 100-kd mitochondrial protein. Immunohistochemical staining using these HmAbs produced mitochondrial staining of septal bile ducts in both PBC and control livers. Ig gene analysis showed that 7 of 13 HmAbs used the V(H)3 and 4 of 13 used VH4 gene repertoire, respectively. Three of 7 V(H)3 antibodies used the same Ig VH3-21 gene family found in human AMA from patients with PBC. The CDRs of these autoantibodies were slightly mutated when compared with the sequences present within the Ig germline genes. In conclusion, the XenoMouse not only recapitulates the unique specificity and restriction of PBC patients, but indicates that the autoantibodies are derived from a restricted clonal selection process. Such data suggest that the original immunogen leads to somatic mutation without subsequent development of determinant spreading.