Akiyoshi Kawamura

Production and Characterization of Monoclonal Strain-Specific Antibodies against Prototype Strains of Rickettsia tsutsugamushi

We have developed 18 hybridoma cell lines which secrete murine monoclonal strain‐specific antibodies to prototype strains of Rickettsia tsutsugamushi: nine anti‐Gilliam, four anti‐Karp and five anti‐Kato antibodies. All the monoclonal antibodies reacted only with their homologous strains in direct and indirect immunofluorescence (IF), or indirect immunoperoxidase (IP) test. By IF and IP tests with the monoclonal antibodies, 22 strains of R. tsutsugamushi, which were newly isolated from mites, field rodents and patients with Tsutsugamushi disease (scrub typhus) in Japan, were all clearly identified as either Gilliam or Karp type. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that the monoclonal antibodies recognized primarily the polypeptides of an apparent molecular weight of 54 to 56 kilodaltons of the homologous rickettsial surface. The monoclonal antibodies produced in the present study should enhance the serotyping and further analytical investigation of the rickettsial antigens since they recognize the strain‐ or type‐specific polypeptides and do not show any cross‐reaction among strains.

Occurrence of high ratio of males after introduction of minocycline in a colony of leptotrombidium fletcheri infected with orientia tsutsugamushi

In colonies of Leptotrombidium fletcheri mites infected with Orientia tsutsugamushi (Ot), the agent of scrub typhus, males rarely appear. In the present study, the development of a high ratio of males was observed after introduction of minocycline (MC). A high dose of MC was injected subcutaneously into a mouse, and by feeding unfed larvae from an infected mite colony on this mouse, the Ot in the mites were successfully killed. Of a total of 130 unfed larvae attached to the mouse, 29 developed into females; of these, 9 laid an average of 112.4 eggs/female. Unfed larvae in the succeeding generations were attached to untreated mice. All adults in the P and F1 generations were females, and males started to appear at the F2 generation. The ratio of males to females was 332:7, 108:13, 263:61 and 71:9 at the F2, F3, F4 and F5 generations, respectively. These data suggest that Ot in the ovary or gonad may suppress the development of males.

Antibody response of athymic nude mice to hapten coupled to thymus-dependent carrier: The effect of hapten density

Abstract The relationship between epitope density on hapten-protein conjugate and thymus dependency of the antibody response was investigated. Dinitrophenyl (DNP) hapten was coupled to thymus-dependent carrier, keyhole limpet hemocyanin (KLH), and the following preparations were utilized: DNP2.5-KLH, DNP8.6-KLH, and DNP26.6-KLH, where subscripts show average number of DNP groups per 100,000 daltons of KLH. Antibody response was examined in athymic nude (nu/nu) mice injected ip with DNP-KLH by assessing DNP-specific plaque-forming cells (PFC) in the spleen. When 50 to 532 μg of DNP-KLH were given ip and PFC were assayed on the 4th day, it was shown that heavily substituted DNP-KLH evoked anti-DNP response to a considerable extent in nu/nu mice at a low dose (50 μg), while lightly substituted one did not even at a high dose (532 μg). The degree of PFC response in nu/nu mice was expressed as percentage of responsiveness in euthymic mice and the following results were obtained by immunizing various antigen preparations: 48.7% in DNP26.6-KLH, 21.3% in DNP8.6-KLH, 11.0% in DNP2.5-KLH. DNP2.5-KLH did not induce a significant antibody response in nu/nu mice at a dose of 532 μg, which was equivalent to 50 μg of DNP26.6-KLH in terms of DNP molecule. These data suggest that large thymus-dependent carrier with repeating arrangement of the epitope (DNP) can directly trigger bone marrow-derived lymphocytes.

Methodological Aspects of Immunofluorescence Applied to Nephrology

The influences of different conditions of specimens and conjugates, especially of pretreatments on the fluorescent patterns of kidneys with positive staining for immunoglobulins were examined. The nature of immunoglobulins might be differentiated by the proper combination of pretreatments. It was also found that fluorescent patterns of kidneys were remarkably changed by different methodological conditions.

Effect of Molar Ratio of Fluorescent Dye to IgG on the Detection of Lymphocyte Surface Immunoglobulins by Immunofluorescence

Labeled antibodies with different F/P molar ratios of FITC to protein (F/P molar ratio) were used for the detection of surface immunoglobulin (S‐Ig) of human and mouse lymphocytes by membrane immunofluorescence, and the following results were obtained. The percentage of S‐Ig bearing cells increased markedly when labeled anti‐human H‐ or L‐chains antibodies were used with higher F/P molar ratios. The investigation of frozen kidney sections of mice injected with human immunoglobulin revealed that such an increase of the positive ratio in S‐Ig was caused by increased non‐specific adsorption of the fraction of labeled antibody with a high F/P molar ratio. This non‐specific adsorption phenomenon was observed at various intensities in materials from different species; materials from mice showed less non‐specific adsorption than those from humans. It was possible to exclude reactivity with an Fc receptor using the top one third of the supernatant of labeled antibody centrifuged at 150,000 for 30 min.

The rescue of Epstein-Barr virus from primary cultured cells of nasopharyngeal carcinoma

Epstein-Barr virus (EBV) was rescued from biopsy cultures of nasopharyngeal carcinoma (NPC) directly after co-culture with foetal cord blood lymphocytes (Cord-Ly). Co-culture with Cord-Ly was performed on 6 NPC, 2 non-malignant lymphoproliferation (NP-N) and 5 lymphoma (NP-L) of the nasopharynx. In 2 out of 6 samples of NPC biopsy, lymphoblastoid cell lines which expressed EBV-determined nuclear antigen (EBNA) by immunofluorescence have been established from co-cultured Cord-Ly of opposite sex to origin, and similarly a cell line has also been obtained from a continuous culture during one month. One out of 2 cell lines which carried EB viral capsid antigen (VCA) has contained EBV particles. One EBNA positive cell line was obtained from NP-N, but it consisted of mixed cells derived from donor and original tumour. One cell line out of 5 NP-L was established, however, it had no evidence for EBV related tests. These findings suggested biological activity of EBV in NPC tumour cells in vitro.