Norio Yumoto

Identification of Annexin 1 as a Novel Autoantigen in Acute Exacerbation of Idiopathic Pulmonary Fibrosis

Consistent with the hypothesis that pulmonary epithelial apoptosis is the key to the acute exacerbation of idiopathic pulmonary fibrosis (IPF), we conducted serological identification of Ags by recombinant expression cloning (SEREX) analysis using type II alveolar cell carcinoma (A549) cell lines to identify disease-related Abs. In a survey of Abs to the recombinant autoantigens identified by SEREX analysis, five Abs were identified as novel candidates for the acute exacerbation of IPF. Abs to annexin 1 were detected in 47 and 53% of the sera and bronchoalveolar lavage materials from patients with acute exacerbation of IPF. Some identical TCR Vβ genes were identified in sequential materials obtained at 1–3 mo in all 10 acute exacerbation IPF cases, suggesting that some infiltrating CD4-positive T cells sharing limited epitopes expand by Ag-driven stimulation during disease extension. The CDR3 region of these identical TCR Vβ genes showed high homology with the N-terminal portion of annexin 1, including in the HLA-DR ligand epitopes predicted by TEPITOPE analysis. By Western blotting analysis and observation of the CD4-positive T cell responses in bronchoalveolar lavage samples, the N-terminal portion of annexin 1 was cleaved and found to induce marked proliferative responses of CD4-positive T cells in three patients. Our study demonstrates that annexin 1 is an autoantigen that raises both Ab production and T cell response in patients with acute exacerbation of IPF, and that the N-terminal portion of annexin 1 plays some role in the pathogenesis of acute exacerbation in IPF patients.

Epstein-Barr virus genomes in Hodgkin's disease and non-Hodgkin's lymphomas

Seventeen of 40 cases of Hodgkins disease (HD) and eight of 46 non‐Hodgkins lymphomas (NHL) were associated with Epstein‐Barr virus (EBV) infection, judged by the EBER‐1 in situ hybridization (ISH) method. Approximately 40% incidence in HD was comparable to previous reports. Young children and elderly HD patients were more prone to be found EBV positive. Fourteen of 17 HD and two of 8 NHL cases with positive EBER‐1 ISH were also positive on LMP‐1 immunostaining. EBV might have a role in lymph‐omagenesis in these cases. The fact that 7 of 8 EBV‐related NHL were peripheral T cell lymphoma indicates the necessity of a larger‐scale survey on this subject. As the present study revealed four cases with positive LMP‐1 immunostaining but negative EBER‐1 ISH (1 HD, 3 NHL), LMP‐1 alone should not be regarded as a tool to prove EBV infection.

Monoclonality of B-cell lineage in primary pulmonary lymphoma demonstrated by immunoglobulin heavy chain gene sequence analysis of histologically non-definitive transbronchial biopsy specimens

Immunoglobulin heavy chain (IgH) gene rearrangements were amplified in transbronchial lung biopsy (TBLB) specimens taken from five patients with primary lymphoma of the lung in whom the diagnosis was established by surgical specimens. By histopathological analysis of TBLB specimens, only two of the five cases were diagnosed as lymphoma, the other three cases being classified as equivocal due mainly to low levels of cellular atypia and to artefactual distortion. All five TBLB specimens, as well as the subsequent surgical specimens, showed a sharp monoclonal band of IgH gene rearrangement on electrophoresis of polymerase chain reaction (PCR) products. By contrast, three surgical biopsy specimens from cases of lymphoid interstitial pneumonia (LIP) showed smear polyclonal bands. No clonal rearrangements were detected in six non‐neoplastic controls, including five cases of chronic bronchitis and one of sarcoidosis. The PCR products of three of the lymphoma cases were sequenced from both TBLB and surgical specimens. In all three cases, there was dominant expression of a particular rearrangement, assumed to be tumour‐derived. In each case, the major clones derived from the TBLB and the surgical specimen were identical. In both lymphoma and LIP cases, more frequent usages of JH4 and JH6 were evident. The diagnosis of lymphoma can be confirmed on TBLB specimens by use of this technique.

CDR3 Sequences of MALT Lymphoma Show Homology with Those of Autoreactive B‐Cell Lines

We have examined the CDR3 sequence and adjacent regions of immunoglobulin genes from B‐cell lymphoma of mucosa‐associated lymphoid tissue (MALT). Twenty‐nine sequences (15 sequences from 13 low‐grade MALT lymphomas, marginal zone B‐cell lymphomas; 7 sequences from 6 highgrade MALT lymphomas; 7 sequences from 7 diffuse large cell lymphomas) were obtained after cloning of the polymerase chain reaction‐amplified segments. In the low‐grade MALT, high‐grade MALT and diffuse large cell lymphomas, the mean length of the CDR3 region was 47.6 ± 10.31 (range 21 to 60), 38.71 ± 10.37 (range 27 to 57) and 40.86 ± 3.34 (range 39 to 48) nucleotides, respectively. The length of the CDR3 region was significantly greater in the low‐grade MALT lymphoma group than in the other two groups. CDR3 sequences in lymphoma cell clones of 14 cases showed 60 to 81% homology with autoantibody‐associated lymphocyte clones including rheumatoid factor. The incidences of these autoantibody‐associated lymphocyte clones were higher in the high‐grade MALT (4/6) and diffuse large lymphomas (5/7) than in the low‐grade MALT lymphoma (5/13). Cases with more than 70% homology at the nucleotide level were found to have 71 to 82% homology with autoantibodies at the protein level in the low‐grade MALT lymphomas (2/13), and 67% homology in the high‐grade MALT lymphomas (2/7). These results indicate that MALT lymphomas may be derived from the malignant transformation of autoreactive B‐cells.

Reappraisal of the Relationship Between Immunoglobulin Heavy Chain Gene Rearrangement and Epstein-Barr Virus Infection in Reed-Sternberg Cells of Hodgkin's Disease

We investigated 44 cases of Hodgkins disease for Epstein-Barr virus genome with EBER-1 in situ hybridization. Twenty of 44 (45.5%) were positive for EBV. Simultaneously, immunoglobulin gene rearrangements were assessed in 32 of these 44 cases with PCR on DNA extracted from Reed-Sternberg cell (RS-cell) -rich areas microdissected from paraffin sections. Clonally rearranged immunoglobulin (IgH) gene was observed in 15 cases (46.9%). EBV-negative cases showed more frequent IgH rearrangement than EBV-positive cases (10 and 5 cases, respectively). In 9 cases, the RS cells were CD20-positive immunohistochemically and these were all EBV negative and the IgH gene was rearranged in all except one. These findings may suggest that EBV infection has occurred before the immunoglobulin gene rearrangement or that EBV infection has influenced the rearrangement of the immunoglobulin gene. The results may also hint towards the obscure B-cell nature of the RS cells.

Immunoglobulin Gene Analysis of Cutaneous Pseudolymphoma by Polymerase Chain Reaction

Polymerase chain reaction (PCR) was used to amplify the V‐D‐J region of the immunoglobulin heavy chain gene from DNA extracted from the formalin fixed, paraffin embedded skin of 3 cases of pseudolymphoma. The products were electrophoresed and observed under ultraviolet light after ethidium bromide staining. Specimens of two cases showed smears of polyclonal amplified DNA. The specimen of one case (Case 3), however, showed a single band with a smear. The presence of monoclonality in B lymphocyte populations may suggest the possibility of low grade malignancy of pseudolymphoma or transformation to malignant lymphoma in the future.

POTENTIATION OF LARGE INTESTINAL TUMORIGENICITY OF CYCASIN DERIVATIVE BY HIGH–FAT DIET AND LACTOBACILLUS IN GERMFREE MICE

Large intestinal tumorigenesis was investigated in germ free (GF) mice following the administration of methylazoxymethanol (MAM) acetate, lard, and lactobacillus arabinosus. Twelve weekly doses of 0.3 mg/10 g MAM acetate given to groups of male weanling, germfree ICR mice, fed either low‐fat (LF) chow alone, or chow with added lard (HF). Additional groups of mice, on the low‐fat diet, were monocontaminated with lactobacillus arabinosus (GA) and they and their GF controls were given 12 weekly doses of 0.2 mg/10 g of MAM acetate. The HF and LF groups were sacrificed after 123 days, and the GA and GF groups after 214 days. Serial sections of the entire large intestine of the MAM acetate‐treated mice revealed many more sessile polyps in the mice maintained on the high‐fat diet. In the mice monocontaminated with lactobacillus, numerous tumors were found, of which 25 percent were invasive. In addition, the polyps found in these animals were generally considerably larger than the polyps found in the GF mice. Atypia occurred in the large intestinal mucosa in all groups. Lard enhanced the large intestinal polypogenesis induced by MAM acetate in germfree mice. Gnotobiosis potentiated the formation of sessile polyps, and carcinogenesis.

BCL-6 mutations in pulmonary lymphoproliferative disorders: demonstration of an aberrant immunological reaction in HIV-related lymphoid interstitial pneumonia.

We used a PCR and sequence procedure to analyze the Ig VH gene and the mutations in the 5′ regulatory regions of BCL-6 genes in pulmonary lymphoproliferative disorders (mucosa-associated lymphoid tissue (MALT) lymphoma, HIV-related, EBV-related, and virus-negative lymphocytic interstitial pneumonia (LIP)). Eight of 20 (40%) pulmonary MALT lymphoma and 10 of 20 LIP (5 of 5 (100%) HIV-related, 2 of 5 (40%) EBV-related, and 3 of 10 (30%) virus-negative LIP) cases showed BCL-6 gene mutations. Intraclonal heterogeneity of the BCL-6 mutations was observed only in pulmonary MALT lymphoma cases whose Ig VH genes also showed intraclonal heterogeneity. Ongoing BCL-6 mutations might reflect re-entry into a germinal center pathway to further mutations. BCL-6 mutations in pulmonary MALT lymphoma and HIV-negative LIP showed some features (high transition to transversion ratio, standard polarity, and RGYW/WRCY bias) of Ig VH gene hypermutation, leading to the view that pulmonary MALT lymphomas and HIV-negative LIP are under the influence of germinal center hypermutation mechanisms. Because BCL-6 mutations in HIV-related LIP cases did not demonstrate features of Ig VH gene hypermutation, immunological reactions in HIV-related LIP are the result of a process different from that found in HIV-negative pulmonary lymphoproliferative disorders.

Aberrant Expression of Immunoglobulin Heavy Chain Genes in Epstein-Barr Virus-Negative, Human Immunodeficiency Virus-Related Lymphoid Interstitial Pneumonia

The two-step polymerase chain reaction (PCR) and sequencing analysis was used to analyze the immunoglobulin heavy chain variable (Ig VH) genes of open-chest biopsy or autopsy samples from five patients with Epstein-Barr virus-negative human immunodeficiency virus (HIV)-related lymphoid interstitial pneumonia (LIP), and the results were compared with those for Ig VH genes from five HIV-negative LIP patients. The findings of this study are consistent with the different immunological situations of HIV-related and HIV-negative LIP. (a) The Ig VH3 subgroup was underexpressed in three of five cases of HIV-related LIP. In contrast, none of the HIV-negative cases showed this abnormality. Because the Ig VH3 subgroup encodes the largest portion of Ig VH genes, a depletion of B cells expressing Ig VH3 genes reflects a major alteration in the B-cell compartment. (b) All HIV-related LIP cases demonstrated two or three oligoclonal populations. HIV-negative cases showed minor monoclonal or polyclonal populations, but not oligoclonal ones. These oligoclonal populations suggest the coexistence of several occult clonal B-cell populations in HIV-related LIP. (c) Some oligoclonal clones in HIV-related LIP showed mutated framework regions not demonstrated in HIV-negative clones. This degree of variation exceeds the usual mutation rate for frameworks, suggesting a role for framework residues in antigen binding. (d) The frequency of D-D fusions of minor oligoclonal clones (HIV-related LIP) is higher than that of minor monoclonal clones (HIV-negative LIP). Such D-D fusions may enhance the probability of expression of antibodies capable of binding HIV glycoproteins.

T CELL-RICH B CELL LYMPHOMA BEARING EPSTEIN-BARR VIRUS IN TUMOR CELLS : A CASE OF IBL-T-LIKE LESION FOLLOWING LENNERT'S LESION

A case of T cell‐rich B cell lymphoma (TCRBCL) with Epstein‐Barr virus (EBV) infection in tumor cells is reported. A 50 year old male developed right cervical lymph node swelling in July 1988. Initial biopsy in April 1989 demonstrated many scattered Hodgkinoid atypical cells with Lennerts lesion. After partial remission following chemotherapy, the lymph nodes enlarged again, and a second biopsy in February 1991 showed an IBL‐T‐like lesion. Only a small number of Hodgkinoid atypical cells were still observed. After apparently, complete remission, the lesion soon recurred and the patient died in November 1992. Immunohistochemically the Hodgkinoid cells were positive for L26, but negative for LN2, LN3, UCHL‐1, MT1, lysozyme, Ber‐H2 and Leu‐M1. Reactivity for immunoglobulins showed falsepositive because of poly‐clonal staining. IgH monoclonality was detected by the poly‐merase chain reaction method in the first biopsied specimen, and by Southern blotting in the second biopsied snap‐frozen specimen. Monoclonal TCRβ rearrangement was not detected. The Hodgkinoid atypical cells were positive for EBVencoding RNA by in situ hybridization, and LMP‐1 by immunostaining. Occasionally, EBV‐bearing immunoblastic, medium sized, or small lymphocytic cells were also observed. This case indicates the possibility that EBV is related to the pathogenesis of TCRBCL.