Characteristics of murine non-specific killer cells induced in vivo by recombinant human interleukin-2.

We have examined the induction of murine non-specific killer cells in vivo and in vitro by purified recombinant human interleukin-2 (rIL-2), and compared their characteristics with respect to killing ability, cell surface phenotypes, and antibody-dependent cell-mediated cytotoxicity (ADCC). C57BL/6 spleen cells cultured with rIL-2 were remarkably cytotoxic against a variety of tumour cells in a 4-hr 51Cr-release assay. Treatment with various antibodies (anti-Thy 1, anti-Lyt 1, anti-Lyt 2, and anti-asialo GM1) plus complement (C) showed that anti-Thy 1 or anti-asialo GM1 antibody plus C removed a majority of killer activity (80% and 66%, respectively). In addition, an increase in ADCC was detected in the spleen cells cultured with rIL-2. These ADCC effector cells were indistinguishable from non-specific killer cells by the cell surface phenotypes. A single administration of rIL-2 in vivo induced only transient and marginal enhancement of non-specific killer activity of spleen cells in C57BL/6 mice. On the other hand, when 10 micrograms of rIL-2 were administered daily by bolus to C57BL/6 mice, the activity increased gradually for about 10 days and reached a plateau. This enhanced non-specific killer activity rapidly decreased and returned to normal by 72 hr after the administration was stopped. The non-specific killer cells induced in vivo in this manner were not only greatly cytotoxic against natural killer (NK)-sensitive tumour cells but were also significantly cytotoxic against NK-resistant tumour cells. Most of the killer activity (more than 90%) was specifically removed by treatment with anti-Thy 1 or anti-asialo GM1 antibody plus C. An increase in ADCC was detected concurrently with an increase in non-specific killer activity in vivo, and both effector cells were indistinguishable by their cell surface phenotypes. These results indicate that a majority of non-specific killer cells induced both in vivo and in vitro by rIL-2 have some common features. Our results also suggest that these cells belong to the same lineage as NK cells, although they are thought to be at different stages from resident NK cells.     

Mutation analysis of two Japanese patients with Fanconi-Bickel syndrome

Fanconi-Bickel syndrome (FBS), or glycogen storage disease type XI, is a rare autosomal recessive disorder characterized by hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Recently, this disease was elucidated to link mutations in the glucose transporter 2 (GLUT2) gene. Only three mutations in three FBS families have been reported. Therefore, it is important to elucidate mutations in the GLUT2 gene in FBS by answering the question of whether the syndrome is a single gene disease. In this report, we describe two patients in two unrelated families clinically diagnosed with FBS. No mutation in the entire protein coding region of the GLUT2 gene was detected in patient 1, which suggested that no mutation existed in the GLUT 2 gene, or that some mutations had affected the expression of the GLUT 2 gene. In patient 2, a novel homozygous nonsense mutation (W420X, Trp at codon 420 to stop codon) was detected. These results support the correlation between GLTU2 gene mutation and FBS syndrome. However, many patients must be analyzed to determine whether other genes are involved in FBS. Received: July 16, 1999 / Accepted: September 3, 1999     

A highly polymorphic CA repeat marker at the human tumor necrosis factor alpha (TNFAα) locus

Tumor necrosis factor alpha (TNFα) in activated monocytes exerts cytotoxic activity and has a variety of other biological effects. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the gene located at 6p21.3. High heterozygosity (0.80) makes this polymorphism a useful marker in the genetic study of disorders affecting immunological response and cell differentiation. Received: June 2, 1998 / Accepted: June 24, 1998     

Localization of sex steroid receptor cells, with special reference to thymulin (FTS)-producing cells in female rat thymus

Using monoclonal antibodies against progestin receptors (PR) and estrogen receptors (ER), and polyclonal antibodies to thymulin (FTS) and keratin, localization of the sex steroid receptors was studied immunohistochemically in ovariectomized estrogen-treated rat thymus, with special reference to FTS-producing cells. Both ER- and PR-immunostained cells were mainly localized in the medullary region, especially at its periphery (i.e., the corticomedullary junction). A few cells were also situated in the subcapsular area. They were medium- to large-sized and had a dendritic cell process, some of which were immunohistochemically keratin- and FTS-positive, indicative of reticuloepithelial (RE) cells. Hassall's corpuscles were also receptor-positive and FTS-positive. T-cells were not immunostained with anti-ER, anti-PR or anti-FTS. Light microscopically, both ER and PR immunostainings were localized in the cytoplasm and/or nucleus of keratin-stained RE cells. Electron microscopically, both steroid receptors were shown more precisely to distribute as aggregates of osmiophilic black dots on polysomes and perinuclear space in the cytoplasm and on the euchromatin area in the nucleus. These results suggest that the sex steroids E and P exert their effects through receptors within RE cells which produce FTS to regulate T-cell differentiation.     

Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese

Molecular diagnosis makes a substantial contribution to precise diagnosis, subclassification, prognosis, and selection of therapy. Mutations in the PDS (SLC26A4) gene are known to be responsible for both Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct, and the molecular confirmation of the PDS gene has become important in the diagnosis of these conditions. In the present study, PDS mutation analysis confirmed that PDS mutations were present and significantly responsible in 90% of Pendred families, and in 78.1% of families with nonsyndromic hearing loss associated with enlarged vestibular aqueduct. Furthermore, variable phenotypic expression by the same combination of mutations indicated that these two conditions are part of a continuous category of disease. Interestingly, the PDS mutation spectrum in Japanese, including the seven novel mutations revealed by this study, is very different from that found in Caucasians. Of the novel mutations detected, 53% were the H723R mutation, suggesting a possible founder effect. Ethnic background is therefore presumably important and should be noted when genetic testing is being performed. The PDS gene mutation spectrum in Japanese may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of a variety of diseases.     

Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.     

Clear cell adenocarcinoma with endobronchial polypoid growth

Clear cell adenocarcinoma of the lung is extremely rare. On radiography, a 45-year-old female with fever was found to have an abnormal shadow in the left lower lung field. Bronchoscopy revealed a polypoid tumor in the left bronchus. On biopsy, the tumor was determined to be adenocarcinoma. Preoperative examination found no tumors outside of the lung. The patient underwent left lower lobectomy with bronchial wedge resection. The tumor had completely obstructed and dilated the left lower bronchus, but had not invaded the tissue outside the bronchial wall. Microscopically, the cytoplasm of the tumor cells contained abundant glycogen, and the tumor had solid and glandular structures. The tumor was diagnosed as clear cell adenocarcinoma of the lung.     

Cisplatin-induced long-term dynorphin A-immunoreactivity in cell somata of rat area postrema neurons

We evaluated long-term dynorphin A-immunoreactivity in the rat area postrema (AP) after the administration of cisplatin. First, rats were given 1, 5 and 10 mg/kg body weight cisplatin (i.p.) and their behavior was monitored for 72 h. We observed a delayed increase in pica 24-72 h after injection, compared to the 24 h before injection. We attributed this to the cisplatin injection. Pica was defined as an increase in the intake of non-nutritional matter such as kaolin. Administration of 1, 5 and 10 mg/kg cisplatin led to an increase in kaolin intake on day 1. Administration of 5 and 10 mg/kg of cisplatin led to decreased intake of laboratory chow (MF) on days 1–3, but 10 mg/kg cisplatin causes an excessive aggravation of their condition. Following this behavioral experiment, we immunohistochemically examined the induction of dynorphin A in the AP at 24, 48 and 72 h post-administration of 1 and 5 mg/kg cisplatin. Administration of 5 mg/kg cisplatin caused dynorphin A to accumulate gradually in the neurosoma of the AP neurons, and the numbers of positive AP neurosomata at 48 and 72 h post-administration were higher than following an equal dosage of 0.9% NaCl. These findings suggest that dynorphin A increases in the central nervous system for a long time following administration, and causes certain behavioral and clinical changes, including those related to appetite and nausea.