Kazuhisa Hasui

Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor β

Tumor-associated macrophages (TAMs) are frequently found in glioblastomas and a high degree of macrophage infiltration is associated with a poor prognosis for glioblastoma patients. However, it is unclear whether TAMs in glioblastomas promote tumor growth. In this study, we found that folate receptor β (FRβ) was expressed on macrophages in human glioblastomas and a rat C6 glioma implanted subcutaneously in nude mice. To target FRβ-expressing TAMs, we produced a recombinant immunotoxin consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRβ monoclonal antibody and Pseudomonas exotoxin A. Injection of the immunotoxin into C6 glioma xenografts in nude mice significantly depleted TAMs and reduced tumor growth. The immunotoxin targeting FRβ-expressing macrophages will provide a therapeutic tool for human glioblastomas.

Analysis of p53, K‐ras, c‐kit, and β‐catenin gene mutations in sinonasal NK/T cell lymphoma in northeast district of China

Recently we reported the different frequenties of p53 and c‐kit gene mutations among sinonasal NK/T cell lymphoma (NKTCL) in Korea, north China (Beijing), and Japan, suggesting some racial, environmental, or life‐style differences as a possible cause of nasal tumorigenesis. In this study, gene mutations in p53, c‐kit, K‐ras, and β‐catenin gene were analyzed by polymerase chain reaction (PCR)‐single strand conformation polymorphism (SSCP) followed by direct sequencing in 20 cases of sinonasal NKTCL from northeast China (Shen Yang). Age of patients ranged from 5 to 63 (median, 40.0) years. p53 gene mutations were found in eight of 20 cases (40%), with exon 4 involvement in 10% of cases. The majority was missense mutations and G:C to A:T transition was predominant. The frequency of the c‐kit and K‐ras gene mutations was low (5%), while that of the β‐catenin gene was six of 20 cases (30%). From these findings, it is concluded that nasal NKTCL in northeast China shared common features with that in Korea in the younger onset of disease compared to that in Japan and lower frequency of p53 gene mutations with infrequent exon 4 involvement compared to that in Japan and north China. These differences might be caused by migration of susceptible populations or some environmental confounding factors. (Cancer Sci 2003; 94: 297–301)

Effect of an immunotoxin to folate receptor β on bleomycin-induced experimental pulmonary fibrosis

It has been suggested that alveolar and interstitial macrophages play a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) by producing proinflammatory and/or fibrogenic cytokines. We showed that inflammatory macrophages expressed folate receptor β (FRβ) while resident macrophages in normal tissues expressed no or low levels of FRβ. In the present study, we examined the distribution of FRβ‐expressing macrophages in the lungs of patients with usual idiopathic pulmonary fibrosis (UIP) and mice with bleomycin‐induced pulmonary fibrosis (PF) and tested whether the depletion of FRβ‐expressing macrophages could suppress bleomycin‐induced PF in mice. Immunostaining with anti‐human or ‐mouse FRβ monoclonal antibody (mAb) revealed that FRβ‐expressing macrophages were present predominantly in fibrotic areas of the lungs of patients with UIP and mice with bleomycin‐induced PF. Intranasal administration of a recombinant immunotoxin, consisting of immunoglobulin heavy and light chain Fv portions of an anti‐mouse FRβ mAb and truncated Pseudomonas exotoxin A, increased survival significantly and reduced levels of total hydroxyproline and fibrosis in bleomycin‐induced PF. In immunohistochemical analysis, decreased numbers of tumour necrosis factor‐α‐, chemokines CCL2‐ and CCL12‐producing cells were observed in the immunotoxin‐treated group. These findings suggest a pathogenic role of FRβ‐expressing macrophages in IPF. Thus, targeting FRβ‐expressing macrophages may be a promising treatment of IPF.

Expansion of a unique macrophage subset in rheumatoid arthritis synovial lining layer

The Z39Ig protein (complement receptor for C3b and iC3b) is expressed on resident tissue macrophages in various tissues. This study was undertaken to examine the distribution of Z39Ig+cells and their phenotypic features in rheumatoid arthritis (RA) synovium, in comparison with those of osteoarthritis (OA) and psoriatic arthritis (PsA) synovium. Monoclonal anti‐Z39Ig antibody was produced by immunizing Z39Ig transfected murine pre B cells and used for the identification of Z39Ig+cells. Z39Ig+cells were further stained with antibodies to macrophages, fibroblast‐like synoviocytes, complement receptors and dendritic cells by using the double immunostaining method in normal, RA, OA and PsA synovium. RA synovial mononuclear cells were double‐stained using anti‐Z39Ig and anti‐CD11c antibodies and sorted into Z39Ig+CD11c+cells and Z39Ig+CD11c−cells. These cell populations were then analysed by electron microscopy. The expression of the Z39Ig protein was limited to intimal macrophages in normal, RA, OA and PsA synovium. The numbers of Z39Ig+CD11c+cells and the ratios of Z39Ig+CD11c+cells to Z39Ig+cells were increased in the synovial lining layer of RA as compared with those of OA and PsA. The ultrastructural analysis of Z39Ig+CD11c+cells showed the character of macrophages with many secondary lysosomes and swelling of mitochondria. Z39Ig+ cells appeared to be useful for identification of resident tissue macrophages in normal synovium and the corresponding macrophages in the synovial lining layer of inflammatory arthritis. Expansion of Z39Ig+CD11c+cells was characteristic of RA synovial lining layer.

Double Autoimmunostaining with Glycine Treatment

Double autoimmunostaining by a sequential twice-repeated enzyme-labeled polymer method was examined on archival paraffin sections of formalin-fixed human tissue using an autoimmunostaining apparatus to determine optimal conditions for glycine treatment, to select the best combination of dyes for the horseradish peroxidase–hydrogen peroxide reaction, and to investigate mounting methods for preparing permanent specimens. The optimal glycine treatment determined by changing the incubation time in 0.1 M glycine hydrochloride buffer, pH 2.2, was glycine buffer washing three times for 1 min each, with suppression of nonspecific binding of the primary antibody by protein blocking. Combinations of DAB and AEC, SG and AEC with Ultramount, and DAB and VIP or No-vaRED and SG with the VectaMount were found usable for the double autoimmunostaining, based on color analysis of the dyes. Pairs of primary antibodies, CD68 and anti-fascin antibodies CD3 and CD79a, and anti-Ki-67 antigen and anti-p53 antibodies were applicable in double autoimmunostaining with appropriate antigen retrieval for each pair of primary antibodies. Consequently, good sequential double autoimmunostaining should include masking the nonspecific binding of primary antibodies, optimal glycine treatment, and selection of adequate dyes and mounting methods.

Adult T‐cell leukemia/lymphoma in Jujuy, north‐west Argentina

Human T‐cell leukemia virus type 1 (HTLV‐1) infection is prevalent in native Americans living in the Andes. Some of their malignant lymphomas (ML) show a peculiar histology suggestive of adult T‐cell leukemia/lymphoma (ATLL). To determine whether ML resembling ATLL are indeed ATLL, re‐analysis of 34 cases occurring in Jujuy, a province of Argentina, was conducted, concentrating on immunological phenotype, integration of HTLV‐1 proviral DNA, expression of HTLV‐1 p40Tax and p27Rex, and infection of Epstein–Barr virus (EBV). The ML were 22 cases of mature peripheral T‐cell and natural killer (NK)‐cell neoplasm (mT/NKN), 11 B‐cell malignant neoplasms and one Hodgkins lymphoma. Polymerase chain reaction against the HTLV‐1 proviral DNA, using DNA extracted from paraffin sections, indicated integration of the HTLV‐1 proviral DNA in three cases of eight mT/NKN. Two other cases of mT/NKN were positive for anti‐HTLV‐1 antibodies. Expression of p40Tax and p27Rex was detected in all five of these mT/NKN cases associated with HTLV‐1. As such, these five mT/NKN were rediagnosed as ATLL. In situ hybridization signals for EBV‐encoded small nuclear early region‐1 were detected in nine cases of mT/NKN, of which five cases of NK‐cell lymphoma were found to have cytoplasmic CD3 expression, a CD56 phenotype and positivity of TIA1. According to the new World Health Organization classification, the mT/NKN class includes five cases of ATLL and five cases of NK‐cell lymphomas. The five cases of ATLL were of native American extraction from an HTLV‐1‐endemic area around Jujuy, north‐west Argentina.

Immunohistochemical analysis of pericryptal fibroblast sheath and proliferating epithelial cells in human colorectal adenomas and carcinomas with adenoma components

In order to examine stromal–epithelial interaction during the oncogenic progression of large bowel tumors, the association between pericryptal fibroblast sheath (PCFS) and expression of Ki‐67 antigen was evaluated in 87 cases of colorectal adenoma and 95 cases of carcinoma with an adenoma component (CWA). For the immunohistochemistry, anti‐α‐smooth muscle actin antibody (α‐SMA) and anti‐Ki‐67 antigen antibody (MIB‐1) were used. In adenomas and adenoma components of CWA, the quantity of neoplastic glands with PCFS was reduced relative to the progression of histological atypia. Pericryptal fibroblast sheath was virtually absent from invasive carcinoma areas of CWA. Increased expression of Ki‐67 correlated with the degree of histological atypia of adenomas. A significant reverse correlation was also seen between Ki‐67 expression and PCFS‐positive glands in adenoma components of CWA. These findings suggest that the prevalence of PCFS and Ki‐67 expression are important indicators of colorectal neoplasia progression. The significant reduction of PCFS in colorectal epithelial neoplasms reflects progression in the adenoma–carcinoma sequence.

Improvement of supersensitive immunohistochemistry with an autostainer: a simplified catalysed signal amplification system.

The ImmunoMax/catalysed signal amplification (CSA) system is a supersensitive method of paraffin immunohistochemistry. It incorporates antigen retrieval, the streptavidin–biotin complex (sABC) method, and the catalysing reporter deposition/catalysing biotinylated tyramide reaction. Strong, non-specific cytoplasmic reaction in the ImmunoMax/CSA is due to endogenous biotin unmasked in the antigen retrieval step. We examined procedures to diminish this non-specific immunoreaction and improved the ImmunoMax/CSA. Antigen retrieval in a hot water bath yielded a smaller endogenous biotin immunoreaction than antigen unmasking in an autoclave. Post-antigen retrieval fixation in buffered 10% formalin solution suppressed the biotin immunoreaction but masked the target antigen, Ki67. Post-reaction washing with 0.1% Tween 20 in Tris–HCl buffer at 35°C did not diminish the endogenous biotin immunoreaction. Animal serum also did not suppress the non-specific immunoreactivity of biotin and antibodies. Because endogenous biotin is detected by duplicated biotin–streptavidin reactions in the ImmunoMax/CSA, we replaced the sABC step with a labelled polymer secondary antibody (the EnVision system) – a simplified CSA system – because the sensitivity ofx the EnVision system was the same as that of the sABC method. The non-specific immunoreaction induced by the EnVision system was masked competitively by blocking protein. By using an antibody against Ki67 antigen that can react only with the nucleus, we were able to evaluate the non-specific cytoplasmic immunoreaction induced by the detection system. We believe that the simplified CSA system will open up the field of supersensitive paraffin immunohistochemistry.

Inflammatory pseudotumor of the spleen: Report of a case

A case of an inflammatory pseudotumor arising in the spleen of a 60-year-old Japanese male is described herein. This benign lesion is extremely rare, with only 12 cases, including our own, having been reported in the world literature. We preoperatively diagnosed the splenic tumor as a metastasis, due to the coexistance of advanced stage carcinoma in the sigmoid colon. However, after splenectomy, histopathological examination of the mass revealed aninflammatory process. Inflammatory pseudotumors often pose diagnostic difficulties because the clinical and radiologic findings are suggestive of malignancy. The clinical and pathological features of cases previously reported are reviewed following the presentation of this case.

Efficacy of an immunotoxin to folate receptor beta in the intra-articular treatment of antigen-induced arthritis

IntroductionWe previously demonstrated that synovial sublining macrophages express folate receptor beta (FRβ). The aim of this study was to evaluate the efficacy of intra-articular administration of a recombinant immunotoxin to FRβ for treating rat antigen-induced arthritis.MethodsA monoclonal antibody (mAb) to rat FRβ was produced by immunizing mice with B300-19 cells (murine pre-B cells) transfected with the rat FRβ gene. Recombinant immunotoxin was prepared by conjugating the Fv portion of the anti-rat FRβ mAb heavy chain with a truncated Pseudomonas exotoxin A and the Fv portion of the anti-rat FRβ mAb light chain. Antigen-induced arthritis was induced through intra-articular injection of methylated bovine serum albumin (mBSA) after two subcutaneous injections of mBSA and complete Freunds adjuvant. Immunotoxin was intra-articularly injected into the arthritis joint every other day for seven days after arthritis onset. Joint swelling was measured and histological scores of inflammation, synovial thickness, cartilage, and bone destruction were determined. Immunohistochemistry was performed to detect osteoclast and osteoclast precursor FRβ-expressing macrophages and cathepsin K-positive cells on day 21.ResultsIntra-articular administration of the immunotoxin attenuated joint swelling (61% suppression; P < 0.01 compared to the control on day 21) and improved histological findings, particularly cartilage and bone destruction (scores of rats treated with control versus the immunotoxin: 2.2 versus 0.5; P < 0.01), by reducing the number of FRβ-expressing macrophages and cathepsin K-positive cells.ConclusionsIntra-articular administration of an immunotoxin to FRβ is effective for improving rat antigen-induced arthritis.