Monoclonal Antibody 4A4 Research Articles

Factor VIII C2 Domain Epitopes Recognized By Classical and Non-Classical Anti-C2 Domain Monoclonal Antibodies Help Mediate FVIII Uptake By Dendritic Cells

Introduction: Dendritic cells are the predominant antigen presenting cells (APCs) responsible for recognition and processing of pathogens and other proteins to induce a primary immune response. Factor VIII (FVIII) is a highly immunogenic protein and the formation of anti-FVIII antibodies remains the most common complication of hemophilia treatment. The A2 and C2 domains of FVIII account for a significant portion of antibodies formed in patients with severe hemophilia A. Prior studies have shown that C1 domain epitopes mediate FVIII uptake by human and murine monocyte-derived dendritic cells (MDDCs). In contrast, in-vitro uptake of FVIII bound by a human-derived anti-A2 monoclonal antibody (MAb) directed against epitopes Arg484-Ile508 did not alter FVIII intake, suggesting that interactive residues within the A2 domain do not participate in FVIII presentation to APCs. However, in light of the functional and structural similarities between the C1 and C2 domains and current knowledge of the immunogenic B cell epitopes within the C2 domain, further investigation into the C2 domain's role in FVIII uptake by APCs is essential.Methods:Human peripheral blood mononuclear cells were harvested from healthy donors after written consent. Monocytes were isolated by positive selection using CD14 microbeads. CD14+ monocytes were stimulated and expanded into immature MDDCs in medium containing 1000 U/ml GM-CSF and 800 U/ml IL-4 for 4-6 days. To evaluate FVIII uptake by dendritic cells, MDDCs were incubated with 10 nM DyLight650-conjugated recombinant full length FVIII (DyL650-FVIII) in the absence or presence of a saturating concentration of 80 nM of murine-derived anti-human FVIII MAbs following a 30 minute pre-incubation period of FVIII/MAb for optimal binding at 37°C. MAbs tested were classical anti-C2 MAbs 3D12 and I89, non-classical anti-C2 MAb B9, and anti-A2 MAb 4A4. Cells were subsequently washed, fixed, and analyzed by flow cytometry. Internalization of FVIII and median fluorescence intensity of FVIII uptake by MDDCs in the presence of MAbs were compared to MDDCs exposed to FVIII or serum free medium alone.Results: Both classical anti-C2 MAbs 3D12 and I89 reduced the percentage of FVIII internalized by MDDCs by 68% and 70% respectively compared to MDDCs treated with FVIII alone similar to the decrease seen with anti-C1 MAbs. Classical anti-C2 MAbs potently inhibit the interaction of FVIII with key binding ligands of Von Willebrand factor (VWF) and phospholipids. The proposed binding epitopes of 3D12, a group B MAb, includes Leu2251, Leu2252, and Phe2196. MAb I89 is a group AB MAb and the proposed binding epitope encompasses residues Met2199 and Phe2200. Non-classical anti-C2 MAb B9 reduced FVIII uptake by 31%, which is comparably less than the classical anti-C2 MAbs. B9 is a group BC antibody that inhibits FVIII binding to phospholipids but does not inhibit binding to VWF. Prior studies have shown that Lys2227 is a critical structural epitope for group BC anti-C2 MAbs. Anti-A2 MAb 4A4, a MAb that binds the Asp403-His444 epitope, did not significantly alter FVIII uptake demonstrating similar internalization of FVIII to the FVIII control. Representative histograms of the percentage of internalized FVIII by untreated MDDCs (shaded gray), MDDCs treated with FVIII alone (black solid line), and MDDCs treated with FVIII in the presence of the anti-C2 or A2 MAbs tested (black dotted line) are shown (Figure 1). Observed reductions in FVIII uptake in the presence of the anti-C2 MAbs suggests that the binding sites recognized by these MAbs play a role in FVIII uptake by dendritic cells.Conclusion: Binding epitopes recognized by classical and non-classical anti-C2 MAbs mediate FVIII uptake by dendritic cells similar to anti-C1 MAbs. Better understanding of the specific epitopes that promote FVIII uptake and inhibitor formation could ultimately lead to development of mechanisms for inhibitor prediction and prevention. [Display omitted] DisclosuresMeeks:Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees.

Constitutive Cyclin A1 Expression Impairs Retinoic Acid-Induced Growth Arrest and Differentiation of Myeloid Leukemia Cells

AbstractAbstract 2894Acute promyelocytic leukemia (APL) differentiation syndrome (DS) in all-trans retinoic acid (ATRA) therapy is often associated with increase of leukocyte count (hyperleukocytosis). It suggests deregulated cell proliferation of ATRA-induced APL cells is possibly involved in the development of DS. The molecular mechanism(s) of hyperleukocytosis are unknown. We previously found increased expression of cyclin A1 mRNA were associated with both increase of initial leukocyte count and development of DS in the therapy with ATRA in APL. To clarify role(s) of cyclin A1 in the proliferation and differentiation of myeloid leukemia cells, we generated U937.A1 which is stably transfected with cyclin A1 gene. U937.A1 showed increased expression of cyclin A1 protein compared with U937.C, which carries pCDNA3.1Hisc (2.5 fold, immunoblot). U937.A1 showed increased in vitro colony formation compared with U937.C (1.3 fold).Flow cytometry (FCM) analysis of cells stained with propidium iodide(PI) showed that U937.A1 was characterized with significant decrease in cells in G1 and increase in G2M (P<0.01)(U937.C=G1:70.1±1.1,S:26.5±1.1,G2M:13.8±1.0% and U937.A1=G1:55.5±0.9,S:30.1±2.9,G2M:21.1±0.8%). U937.A1 showed constitutive higher Cdk1 kinase activities compared with U937.C more than 100 times, as measured by ELISA which quantitate enzyme activities by detecting phospholyrated synthetic peptide using monoclonal antibody 4A4. It suggests that inappropriate activation of cyclin A1/Cdk1 complex is associated with altered cell cycle progression of U937.A1 cells. Immunoblot analysis showed that U937.A1 over-expressed checkpoint kinase 1(Chk1) protein compared with U937.C.(1.4 fold)suggesting deregulated G2M checkpoint in U937.A1. Anti-apoptotic protein, bcl2, was increased in U937.A1 cells(1.5 fold).Incubation with 1μ M ATRA for 4 days significantly inhibited cell growth in U937.C (U937.C=44.9±1% and U937.A1=70.8±3%, p<0.01, cell No. with ATRA/without ATRA).ATRA-induced growth inhibition in U937.C was accompanied with significant increase of cells in G1 (U937.C=88.9±0.9% and U937.A1=68.2±0.8%) and decreased in the G2M(U937.C=4.9±0.5% and U937.A1=19.7±1.2%). Expression of both cyclin A2 and cyclin B proteins were markedly down-regulated at days 3 and 4 in U937.C in the culture (U937.C=69.1±1.3%(cyclin A2) and 52.1±2.1%(cyclin B), U937.A1=90.2±1.7%(cyclin A2)and 94.5±2.4%(cyclin B),day4, ATRA/without ATRA) Cdk1 protein expression was also significantly down-regulated in U937.C(U937.C=53.5±2.5% and U937.A1=115±5%, p<0.01, ATRA/without ATRA). In both of U937.C and U937.A1 cells, Cdk1 kinase activities were significantly suppressed by 4 days' culture with 1μ M ATRA,however, the activities in U937.A1 still remained 10 times higher than those in U937.C (U937.C=0.06±0.01/0.25±0.02 and U937.A1=0.19±0.01/1.04±0.04,day4, O.D., ATRA/without ATRA). Incubated with ATRA, expression of Chk1 protein in U937.C cells time-dependently decreased (U937.C=44.3±0.1% and U937.A1=86.1±0.1%, p<0.01, day4. with ATRA/without ATRA) Checkpoint kinase activities, as measured by ELISA using anti-phospho-Cdc25C serine 216 specific antibody, was significantly suppressed by ATRA in U937.C (U937.C=1.25±0.02/2.69±0.09 and U937.A1=3.42±0.06/2.79±0.22,day4, O.D., ATRA/without ATRA). Incubation with Go6976 at 10 μ M, a specific chk1 inhibitor, produced significant cell growth inhibition in U937.A1 in combination with ATRA. The growth inhibition by ATRA plus Go6976 was accompanied with accumulation of cells in G1(79.2±0.9%) and decrease of cells in G2M (7.7%±0.3%)compared with those incubated with ATRA alone, suggesting deregulated chk1 activities are involved in impaired ATRA-induced growth inhibition in U937.A1 cells. ATRA-induced down-regulation of expression of bcl2 protein was significant in U937.C (U937.C=41.8±0.1% and U937.A1=77.8±0.2%, day4, with ATRA/without ATRA). Decreased bcl2 protein was inversely co-related with up-regulation of CD11b induced by ATRA (U937.C=4.3±0.2% and U937.A1=3.9±0.1%, FCM, day4).The present results showed that forced expression of cyclin A1 resulted in impaired ATRA-induced growth suppression and differentiation of U937 cells. It was related with inappropriately activated Chk1-Cdk1 pathway. Deregulated G2M cell cycle checkpoint(S)is a candidate for the therapeutic target in ATRA-induced hyperleukocytois in APL. Disclosures:No relevant conflicts of interest to declare.

P63 immunostaining in destained bronchoscopic cytological specimens

The p53 homologous squamous stem-cell regulatory protein p63 is expressed in squamous carcinomas but is not characteristically detected in small-cell carcinomas (SCCs). A panel of thyroid transcription factor (TTF) 1 and p63 has been shown to be useful in distinguishing SCCs from poorly differentiated squamous carcinoma of the lung (PDSLC) in small biopsies and cytological cell blocks. Because tumor samples frequently are limited to cytological smears, we attempted to detect p63 in destained slides from a spectrum of pulmonary malignancies. Archival alcohol-fixed smears from 60 cases of cytologically diagnosed malignancies in bronchoscopically (n = 59) or fine-needle aspiration-obtained specimens (n = 1) were destained in acid alcohol, postfixed in 10% formalin, subjected to citrate-based antigen retrieval, and immunostained by exposure to anti-p63 monoclonal antibody 4A4, followed by reagents from a streptavidin-biotin immunoperoxidase kit, and diaminobenzidine as the chromogen. Postfixation in 10% formalin was found to be necessary for immunostaining. Normal ciliated and goblet cells were p63 negative, but reserve cells were p63 positive. All cases of squamous-cell carcinoma were positive for p63. Of 10 tumor samples originally diagnosed as SCC, only 6 samples were p63 negative and 4 samples exhibited positive staining. However, proper interpretation of the immunohistochemical (IHC) staining pattern and careful scrutiny of the cytological features and biopsy specimens in three of four cases led us to reclassify three cases into PDSLC. All adenocarcinomas (ACAs; n = 12), large-cell carcinomas (n = 4), and metastatic ACAs (n = 5) were p63 negative. Positive staining was seen in 9/16 tumors designated as non-SCCs; these tumors were not classified further into distinct histological categories.p63 staining in destained slides may be of value in facilitating the differential diagnosis between PDSLC and SCC. Criteria for conservative interpretation of results are discussed and include examination of reserve cells and ciliated cells on the same slide as internal positive and negative controls.

P63 Expression in Lung Carcinoma

p63 is a recently discovered member of the p53 family that has been shown to be important in the development of epithelial tissues. p63 may also play a role in squamous cell carcinomas of the lung, head and neck, and cervix, and its expression is increased in these tumors. The purpose of this study was to investigate the expression of p63 in a broad spectrum of histologic types of lung tumors. A total of 441 cases of primary lung tumors with follow-up data were identified, and the paraffin-embedded tissue blocks were used to construct a duplicate core tissue microarray. After review of the tissue cores, 408 cases, consisting of 123 squamous cell carcinomas, 93 adenocarcinomas, 68 large cell carcinomas, 68 classic carcinoids, 31 atypical carcinoids, 11 large cell neuroendocrine carcinomas, and 14 small cell carcinomas, were adequate for analysis. Immunohistochemistry was performed at 2 different laboratories using monoclonal antibody 4A4 to detect the expression of p63, using different staining protocols. p53 expression was also studied with immunohistochemistry using monoclonal antibody DO-7. Kaplan-Meier curves were plotted to compare the survival of p63-expressing versus nonexpressing tumors. A large proportion of squamous cell carcinomas expressed p63 (96.9%), most showing strong positive nuclear immunoreactivity. Expression in other nonsmall cell lung cancers was also present. Thirty percent of adenocarcinomas and 37% of large cell carcinomas showed p63 expression. In the neuroendocrine tumors, an increasing proportion of tumors stained for p63 as tumor grade increased; 1.9% of classic carcinoids, 30.8% of atypical carcinoids, 50% of large cell neuroendocrine carcinomas, and 76.9% of small cell carcinomas were positive. Approximately half of the positively staining neuroendocrine cases showed strong staining. Expression of p63 was of prognostic significance in neuroendocrine tumors (P < 0.0001), with higher-grade tumors more likely to express p63. Correlation between p63 and p53 expression was not observed (P = 0.18) in nonsmall cell lung cancer; however, a significant correlation between the 2 markers was found in neuroendocrine tumors (P < 0.0001). p63 staining was repeated with a different staining protocol, yielding similar results overall but a lower percentage of positive cases (34.2% vs. 48.4% of tumors positive). In conclusion, p63 expression is consistently expressed in squamous cell carcinoma in the lung, but is also expressed in a subset of adenocarcinomas and large cell carcinomas. Pulmonary neuroendocrine tumors also show p63 staining in some instances, particularly in higher-grade tumors, and the majority of small cell carcinomas are p63-positive. These results suggest that p63 may be involved in oncogenesis in a broader range of tumors than was previously thought.

Immunohistochemical detection of p53 homolog p63 in solid cell nests, papillary thyroid carcinoma, and hashimoto’s thyroiditis: a stem cell hypothesis of papillary carcinoma oncogenesis

Most models suggest that the cell of origin of papillary carcinoma is the mature thyroid follicular epithelial cell. In a recent study, p63 was detected in papillary carcinoma, Hashimoto’s thyroiditis, and in squamoid aggregates and solid cell nests (SCNs), embryonic remnants found sporadically in the fully developed thyroid. In the present study, the relationship between solid cell nests and papillary carcinoma was investigated further. Four-micrometer sections from 88 routinely fixed and processed archival thyroidectomy specimens were pretreated with citric acid pH 6.0 for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained with a streptavidin-biotin kit and diaminobenzidine as chromogen and were counterstained with hematoxylin. Squamoid aggregates or SCNs were noted in 21 specimens. Several morphologic variants of SCNs were found, all of which displayed p63 positivity. These included undifferentiated SCNs and those displaying commitment toward squamoid and ciliated glandular differentiation. Small, morphologically inconspicuous aggregates of p63-positive cells were commonly found in Hashimoto’s thyroiditis. Commitment of p63-positive undifferentiated cells toward thyroid follicular epithelial differentiation was occasionally noted. One SCN variant, also associated with Hashimoto’s thyroiditis, was a floretlike arrangement of p63-positive cells with fusiform nuclei. p63 staining was strong and uniform in some SCNs, but in other SCNs it was compartmentalized and homologous to stem cell-staining patterns in normal squamous or bronchial epithelia. Stem cell-like staining, associated with compartmentalized p63 staining or p63-positive undifferentiated cells, was noted in 7 of 27 papillary carcinomas. p63 immunostaining is a highly sensitive means of detecting SCNs. p63 expression patterns in SCNs and a subset of papillary carcinomas are closely homologous to stem cell-associated p63 staining patterns that have been described elsewhere in squamous and bronchial epithelia. We propose a stem-cell-associated model of papillary carcinoma oncogenesis that suggests that (1) p63-positive embryonal remnants rather than mature follicular cells are the cells of origin of a subset of papillary carcinomas; (2) these p63-positive cells are pluripotent and may stay undifferentiated or undergo benign squamoid or glandular maturation, may undergo thyroid follicular epithelial differentiation, may undergo oncogenic change leading to papillary carcinoma, or may trigger an immune reaction, resulting in lymphoid infiltration and Hashimoto’s thyroiditis; and (3) Hashimoto’s thyroiditis and papillary carcinoma may therefore be linked etiologically, because both disorders may be initiated by the same population of pluripotent p63-positive embryonal stem cell remnants.

Expression of p63 in papillary thyroid carcinoma and in Hashimoto’s thyroiditis: a pathobiologic link?

p63 proteins are p53 homologs that are postulated to regulate squamous stem cell commitment. An immunohistochemical survey of p63 expression in normal thyroid and in reactive, neoplastic, and inflammatory thyroid disorders was performed. Sections from routinely fixed and processed archival thyroidectomy specimens were pretreated with citric acid, pH 6.0, for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained using a streptavidin-biotin kit and diaminobenzidine as a chromagen, and then were counterstained with hematoxylin. The results showed that p63 expression was negative in normal thyroid tissue, nodular goiters, and oncocytic follicular adenomas. Positivity was rare and weak in follicular adenomas. p63-positive foci were commonly found in Hashimoto’s thyroiditis (1 or more foci in 78.8% of cases), but rare in Graves’ disease. Twenty-seven of 33 papillary thyroid carcinomas (81.8%) displayed p63-positive foci. Staining was uncommon in follicular carcinomas and rare in medullary carcinomas. One case of insular carcinoma was p63-positive. All squamoid structures were p63-positive; p63-positive structures morphologically consistent with solid cell nests were also identified. Based on the results of this study, we conclude that p63 is commonly expressed in papillary thyroid carcinoma and in Hashimoto’s thyroiditis. Given the debated association of papillary thyroid carcinoma with Hashimoto’s thyroiditis, it is possible that p63 expression may be a potential pathobiologic link between the two disorders. The finding of p63 in benign squamoid nests supports a possible interrelationship between these structures and both Hashimoto’s thyroiditis and papillary carcinoma. The high percentage of papillary carcinomas with p63-positive foci appears to distinguish papillary carcinoma from other neoplasms originating in the thyroid.

P63 In pulmonary epithelium, pulmonary squamous neoplasms, and other pulmonary tumors

p63 is a p53-homologous nuclear protein that appears to play a crucial role in regulation of stem cell commitment in squamous and other epithelia. In this study, p63 expression was examined in benign lung and in neoplasms of pulmonary origin. Eighty sections from routinely fixed and processed archival bronchoscopic biopsy or lobectomy specimens were pretreated with citric acid (pH 6.0) for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained using a streptavidin-biotin kit and diaminobenzidine as chromagen, and were counterstained with hematoxylin. In normal lung, p63 intensely stained nuclei of bronchial reserve cells but did not stain ciliated cells, alveolar epithelial cells, or nonepithelial cells. The lower strata of squamous metaplastic bronchial epithelium stained positively. All squamous-cell carcinomas stained positively (n = 30). In some well-differentiated carcinomas, staining was found at the periphery of tumor nests but was negative in central zones showing squamous maturation. Poorly differentiated carcinomas showed very high proportions (80% to 100%) of p63-positive nuclei. All small-cell carcinomas were p63 negative (n = 9). Staining of bronchioloalveolar carcinomas (n = 7) and adenocarcinomas (n = 23) was variable: some tumors showed no detectable staining, others showed heterogeneously positive staining. Adenosquamous carcinomas (n = 5) displayed a unique basalar staining pattern. Carcinoid tumors were almost entirely negative (n = 5). We conclude that p63 is expressed in benign bronchial stem cells, in neoplastic cells with either squamous differentiation or squamous differentiating potential, and in a subpopulation of adenocarcinomas. p63 immunostaining may also aid in some histopathologic distinctions, such as in small biopsies where the differential diagnosis is poorly differentiated squamous carcinoma versus small-cell carcinoma. A stem cell biology–based classification system for squamous carcinomas is proposed. (HUM PATHOL 33:921-926. Copyright 2002, Elsevier Science (USA). All rights reserved.

A ligand that Trypanosoma cruzi uses to bind to mammalian cells to initiate infection

We purified a soluble gp83 trans-sialidase (gp83-TSA), from phospholipase C-treated Trypanosoma cruzi trypomastigote membranes, which binds to myoblasts, fibroblasts and macrophages to mediate trypanosome entry. Myoblasts display a single class of receptors for the gp83-TSA present at 4×104 per myoblast with a Kd of 8 nM. Monovalent Fab fragments of the monoclonal antibody 4A4 specific for gp83-TSA inhibit gp83-TSA binding to myoblasts, fibroblasts and macrophages, block the trypanosomes from attaching to and entering these cells and neutralize T. cruzi infection in BALB/c mice. This is the first demonstration that gp83-TSA is a ligand that T. cruzi uses to attach to cells.

Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin.

Although accumulating data reveal patterns of proliferation, migration, and differentiation of neuronal lineage cells in the developing brain, gliogenesis in the brain has not been well elucidated. In the rat brain, vimentin is selectively expressed in radial glia and in their progeny, not in oligodendrocytes or neurons from embryonic day 15 (E15) until postnatal day 15 (P15). Here we examined mitotic radial glial lineage cells in the rat brain E17-P7, using the monoclonal antibody 4A4, which recognizes vimentin phosphorylated by a mitosis-specific kinase, cdc2 kinase. In the neocortex, mainly radial glia in the ventricular zone, but not their progeny, underwent cell division. In contrast, not only radial glia but also various types of radial glial progeny including Bergmann glia continued to proliferate in the cerebellum. Radial glia in the neocortex divided horizontally, obliquely, and vertically against the ventricular surface. The percentage of the vertical division increased with progress in the stage of development, concurrently with the decrease of the population of horizontal divisions. Thus, the monoclonal antibody 4A4 provides an useful tool to label mitotic glia in the developing brain and revealed different patterns of gliogenesis in the neocortex and cerebellum. A possibility is discussed that the dynamics of mitotic orientation observed here may be related to the change of the pattern of gliogenesis during development.

Visualization and function of vimentin phosphorylation by cdc2 kinase during mitosis.

To investigate the role of intermediate filament (IF) protein phosphorylation by cdc2 kinase during mitosis, we developed a monoclonal antibody 4A4 recognizing Ser55-phosphorylated vimentin. Western blotting indicated that this antibody reacted with vimentin phosphorylated by cdc2 kinase but not with non-phosphorylated vimentin or with vimentin phosphorylated by other kinases such as cAMP-dependent protein kinase, protein kinase C, or Ca(2+)-calmodulin-dependent protein kinase II. Immunofluorescence and immunoelectron microscopy showed that vimentin Ser55 residues distributed in the entire cytoplasmic vimentin filament system are phosphorylated when the cells enter mitosis and dephosphorylated in cytokinesis. All cell lines examined showed a similar appearance of immunoreactivity with antibody 4A4. Fractionation of mitotic cell extracts on Mono-Q Sepharose revealed a single peak of vimentin Ser55 kinase activity, and the anti-p34cdc2 antibody reacted with the 34 kDa band in the kinase containing fractions. Vimentin Ser55 kinase activities were nil in the interphase cell extract. Immunofluorescent evidence using antibody 4A4 and biochemical analysis using vimentin Ser55 peptide showed that the degree of disassembly of vimentin filament of various cell types at early mitotic phase correlated well with the amount of mitotically activated cdc2 kinase.

A monoclonal antibody, raised against rat embryo hippocampus, identifies a nuclear protein enriched in hippocampal and other neurons of the adult rat brain

We report characteristics of a monoclonal antibody (MAb) produced by immunizing mice against rat embryo hippocampus, and its cellular distribution in the brains of adult and embryonic rats. This antibody, designated as MAb 4A4, brightly stained granular and pyramidal neurons of the adult rat hippocampus, as well as some cortical neurons. Also, MAb 4A4 labeled granular and Purkinje cells of the cerebellum with less intensity. While glial cells were labeled relatively faintly. At embryonic day 18 in the rat, most brain neurons, primitive neuroepithelium, connective tissues and glia were labeled with this antibody, indicating that the expression of 4A4 antigen is regulated developmentally. The 4A4 antigen appeared to be localized to the nucleus of cells except in choroid plexus in which the focal membrane staining was observed. The nuclear localization of 4A4 antigen was further confirmed by the staining of cultured cell lines with MAb 4A4. Western blot analysis demonstrated a single band of the 4A4 antigen from cultured cells, with an apparent molecular weight of 220 kDa. Both the molecular weight and the distribution of the 4A4 antigen in the embryonic and adult rat brain and cultured cells suggest that this antigen is a novel nonhistone nuclear type, which is preferentially expressed in neurons of the rodent brains and is under developmental regulation.