D2-40 antibody immunoreactivity in developing human brain, brain tumors and cultured neural cells.

D2-40 antibody is raised against an oncofetal antigen, the M2A antigen. It has been used as a marker for lymphatic endothelium as well as mesothelioma and cerebellar hemangioblastoma. We demonstrate here that positive D2-40 immunoreactivity was found in the developing cerebrum, particularly in the germinal matrix layer, immature ependyma, choroid plexus and meninges. In the developing cerebellum, positive D2-40 immunoreactivity was found in the external granular layer particularly of the outer portion and the Purkinje cell layer as well as meninges. Some brain tumors such as anaplastic ependymoma, some medulloblastomas, glioblastoma, pineal germinoma, craniopharyngioma, choroid plexus papilloma, choroid plexus carcinoma, and meningioma showed positive immunoreactivity with D2-40. Therefore, D2-40 antibody is considered a useful marker for research on developing brain and diagnosis of brain tumors, differentiation between choroid plexus carcinoma and metastatic carcinoma. In addition, on cultured human neural cells, D2-40 immunoreactivity was found in nestin-positive neural stem/progenitor cells and neuronal lineage cells. As D2-40 antibody recognizes cell surface antigen M2A, it might be a candidate cell surface marker for isolation of human neural stem cells/neuronal lineage cells in the fluorescence-activated cell sorting technique.     

Epstein-Barr virus-positive Hodgkin/Reed-Sternberg-like B cell in non-hodgkin lymphoma: nucleotide sequence of the amplified immunoglobulin heavy-chain variable region gene by the single-cell polymerase chain reaction technique.

We examined nucleotide sequences of Epstein-Barr virus (EBV)-positive Hodgkin/Reed-Sternberg (HRS)-like B cells in a case of diffuse large B-cell lymphoma (DLBCL) and a case of adult T-cell lymphoma (ATL) for single-cell polymerase chain reaction of the immunoglobulin heavy-chain gene variable region (VH gene). HRS-like B cells were scattered in the area irrelevant to the lymphoma infiltrates of DLBCL and in the lymphoma area of ATL. HRS-like B cells were positive for CD20 and CD30 but negative for CD15. EBV presented in HRS-like B cells in both cases but not in any lymphoma cells. VH genes of five HRS-like B cells analyzed in DLBCL were polyclonal and showed in-frame sequences with 0% to 2.8% somatic mutation frequency. In an ATL, VH genes of five HRS-like B cells analyzed were polyclonal and somatically mutated. Four cells carried in-frame rearrangements with 3.5% to 17.7% mutation frequency. One of the VH genes has a one-codon deletion. From the fifth cell, an out-of-frame rearrangement with an insertion and a deletion was obtained. Thus, we showed polyclonal EBV-positive HRS-like B cells in both DLBCL and ATL and that whereas EBV-positive, HRS-like B cells in DLBCL exhibited unmutated and mutated VH gene, those in ATL were found to have a somatically mutated VH gene with/without deletions and/or insertions. The HRS-like B cells may appear because of active EBV infection in a patient who is immunosuppressed from the primary lymphoma.     

Functional monomers and polymers, 105. Synthesis of poly(β-alanine) from a new type of active amino acid ester

Active β-alanine esters having dipeptide groups, which are capable of forming hydrogen bonds between the side chains, were prepared in the form of hydrogen chloride salts. The polycondensation of these salts was studied in non-polar solvents, in the presence of triethylamine. In the case of the ester with β-alanyl-β-alanine as dipeptide moiety, the polycondensation proceeded smoothly in chloroform solution. To explain the characteristic feature of the polycondensation, an ordered aggregation of the ester molecules is assumed to play an important role. The ester with glycylglycine as dipeptide moiety, however, did not undergo a polycondensation.     

Generation of velogenic Newcastle disease viruses from a nonpathogenic waterfowl isolate by passaging in chickens

A benign Newcastle disease virus (NDV) recently became highly virulent during replication in domestic chickens. It is still unclear whether NDVs circulating among wild waterfowl also have the potential to become highly pathogenic (velogenic) in chickens. To demonstrate experimentally the generation of velogenic NDV from a nonpathogenic waterfowl isolate, we passaged an avirulent goose isolate in chickens. After nine consecutive passages by air-sac inoculation, followed by five passages in chick brain, the virus became highly virulent in chickens, producing a 100% mortality rate, and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence, E-R-Q-E-R/L, which progressed incrementally to a typical virulent type, K-R-Q-K-R/F, during repeated passage in chickens. These results demonstrate that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, have the potential to become velogenic after transmission to and circulation in chicken populations. The results also suggest that chickens provide a mechanism for the selection of virulent viruses from an avirulent background.     

Ultrastructure and synaptic organization of the spinal accessory nucleus of the rat

The accessory nucleus is composed of neurons in the medial column that innervate the sternocleidomastoid muscle, and neurons in the lateral column that innervate the trapezius muscle. We retrogradely labeled these neurons by injection of cholera toxin conjugated horseradish peroxidase into the sternomastoid (SM) or the clavotrapezius (CT) muscles, and investigated fine structure and synaptology of these neurons. Almost all SM and CT motoneurons had the appearance of alpha-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles, Nissl bodies, and a prominent spherical nucleus. More than 60% of the somatic membrane was covered with terminals. The SM motoneurons (34.4 x 52.2 microm, 1,363.1 microm(2) in a section) were slightly larger than the CT motoneurons (32.8 x 54.2 microm, 1,180.8 microm(2)). The average number of axosomatic terminals in a section was 52.2 for the SM, and 54.2 for the CT motoneurons. More than half of them (58.0%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II) with the SM motoneurons, while 57.9% of them contained round vesicles and made asymmetric synaptic contacts (Gray's type I) with the CT motoneurons. A few C-terminals were present on the SM (3.5) and the CT (3.7) motoneurons. About 60% of the axodendritic terminals were Gray's type I in both the SM and the CT motoneurons. A few labeled small motoneurons were also found among the SM and the CT motoneurons. They were small (19.2 x 26.2 microm, 367.0 microm(2)), round cells containing poorly developed organelles with a few axosomatic terminals (9.3). Only 20% of the somatic membrane was covered with the terminals. Thus, these neurons were presumed to be gamma-motoneurons. These results indicate that the motoneurons in the medial and the lateral column of the accessory nucleus have different ultrastructural characteristics.     

Infiltrating eosinophils and eotaxin: their association with idiopathic eosinophilic esophagitis.

BACKGROUND: Idiopathic eosinophilic esophagitis (IEE) is a very rare disease characterized by thickening and eosinophil infiltration of the esophagus. The most potent chemotactic factor for eosinophils is eotaxin, and its pathophysiologic significance in IEE needs to be elucidated. OBJECTIVE: To study the association between eotaxin and IEE. METHODS: We examined eotaxin expression in the esophagus of an IEE patient in comparison to controls by immunohistochemistry using a monoclonal antibody for human eotaxin. We also measured the free eotaxin level and the total (free and bound-form) eotaxin level in blood by enzyme-linked immunosorbent assay before and after the initiation of steroid therapy. RESULTS: Most of the infiltrating eosinophils in the affected esophageal tissue showed immunohistochemical staining with anti-eotaxin antibody. In blood samples, the free eotaxin level was slightly elevated before treatment, whereas the total eotaxin level was within the normal range. Unexpectedly, the total eotaxin level increased dramatically after the initiation of steroid therapy, whereas the increase of free eotaxin was modest. CONCLUSION: Infiltrating eosinophils that express eotaxin and the changes of blood eotaxin levels during steroid therapy suggest that eotaxin may be associated with IEE.     

Isolation, characterization, and disruption of dnr1, the areA/nit-2-like nitrogen regulatory gene of the zoophilic dermatophyte, Microsporum canis.

A homolog of the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa was isolated from the zoophilic dermatophyte, Microsporum canis. This gene, dnr1, encodes a polypeptide of 761 amino acid residues containing a single zinc-finger DNA-binding domain, which is almost identical in amino acid sequence to the zinc-finger domains of AREA and NIT-2. The functional equivalence of dnr1 to areA was demonstrated by complementation of an areA loss-of-function mutant of A. nidulans with dnr1 cDNA. To further characterize this gene, dnr1 was disrupted by gene replacement based on homologous recombination. Of 100 transformants analyzed, two showed the results expected for replacement of dnr1. The growth properties of the two dnr1(-) mutant strains on various nitrogen sources were examined. Unlike the A. nidulansareA(-) mutant, these dnr1(-) mutants showed significantly reduced growth on ammonia, a preferred nitrogen source for fungi. These mutant strains were also able to utilize various amino acids for growth. In comparison with wild-type M. canis, the two dnr1(-) mutants showed reduced growth on medium containing keratin as the sole nitrogen source. This is the first report describing successful production of targeted gene-disrupted mutants by homologous recombination and their phenotypic analysis in dermatophytes.     

A Comparative Analysis of the Murine Thymic Microenvironment in Normal,Autoimmune, and Immunodeficiency States

It is widely accepted that the thymic microenvironment regulates normal thymopoiesis through a highly coordinated and complex series of cellular and cytokine interactions. A direct corollary of this is that abnormalities within the microenvironment could be of etiologic significance in T-cell-based diseases. Our laboratory has developed a large panel of monoclonal antibodies (mAbs) that react specifically with epithelial or nonepithelial markers in the thymus. We have taken advantage of these reagents to characterize the thymic microenvironment of several genetic strains of mice, including BALB/cJ, C57BL/6J, NZB/BlnJ, SM/J, NOD/Ltz, NOD/Ltz-scid/sz, C57BL/6J-Hcph m^e/Hcph m^e, and ALY/NscJcl-aly/aly mice, and littermate control animals. We report herein that control mice, including strains of several backgrounds, have a very consistent phenotypic profile with this panel of monoclonal antibodies, including reactivity with thymic epithelial cells in the cortex, the medulla and the corticomedullary junction, and the extracellular matrix. In contrast, the disease-prone strains studied have unique, abnormal staining of thymic cortex and medulla at both the structural and cellular levels. These phenotypic data suggest that abnormalities in interactions between developing thymocytes and stromal cells characterize disease-prone mice.     

A case of a placental site trophoblastic tumor

A rare placental site trophoblastic tumor (PSTT) in a 39-year-old female was studied. This tumor, protruding into the uterine cavity, was histologically similar to tumors in previously reported cases of PSTT. Ultrastructurally, the characteristic finding was the presence of perinuclear filaments. Also, the tumor cells were strongly positive for hPL by immunohistochemical method. These findings suggest that this was a tumor caused by neoplastic proliferation of the extravillous intermediate trophoblast.     

Functional analysis of the effect of forced activation of STAT3 on M1 mouse leukemia cells

M1 mouse myeloid leukemia cells exhibit growth arrest and differentiation to monocytes/macrophages in response to leukemia inhibitory factor (LIF) stimulation. Although recent studies have demonstrated that STAT3 plays a central role in this process, it is unknown whether STAT3 activation alone is sufficient. To address this issue, we have established M1/STAT3ER cells, where STAT3 is selectively activated by 4-hydroxytamoxifen (4HT). 4HT stimulation did not have any effect on growth and morphology of M1/ STAT3ER cells, and did not induce the down-regulation of mRNA of c-myc and c-myb, which is necessary for M1 cell differentiation. On the other hand, mRNA of jun-B, IRF1 and p19 was increased by 4HT. DNA precipitation assay indicated that both stimulation of LIF and 4HT similarly activated STAT3ER. Introduction of a constitutive active MAP kinase kinase (MEK1) into M1/STAT3ER cells did not induce differentiation either. Together, our present data suggest that signaling other than the activation of STAT3 and MEK1 may be necessary for M1 cell-growth arrest and differentiation, while a set of early genes of LIF are induced by only STAT3 activation.