Gelatin beads as platforms for targeting molecule and anti-Fas antibody: two major properties of cytotoxic T lymphocytes.

OBJECTIVE: Cytotoxic T lymphocytes (CTL) initially recognize target cells using the T-cell receptor (TCR), then strongly adhere to these cells by accessory molecules, and finally induce apoptosis by Fas ligand (FasL)/Fas or lyse by the granzyme/perforin system. We describe the development of gelatin beads carrying anti-tumor monoclonal antibody (mAb) and anti-Fas mAb mimicking the TCR and FasL, respectively. We hypothesized that these antibody-coated beads can be therapeutically utilized for the elimination of tumor cells. MATERIALS AND METHODS: We evaluated the cytotoxic activity of gelatin beads bearing CH11 (anti-Fas mAb) after incubation with several human leukemia cell lines. Cytotoxic activities were measured using colorimetric DNA fragmentation assay and lactate dehydrogenase (LDH) release assay. RESULTS: We demonstrated that the cytotoxic effects of anti-Fas mAb were markedly enhanced by fixation on gelatin beads. Microscopic examination showed that the beads attached to the target cells and induced their apoptosis. These effects were enhanced further by adding tumor-specific mAb. These in vitro properties of the beads were well reconstituted in the peritoneal cavity of mice. CONCLUSION: Although antibody-coated gelatin beads lack several important properties of natural CTL, such as differentiation, proliferation, and the functions of adhesion molecules, they mimic well the targeting and cytotoxic functions of natural CTL. Our findings suggest that antibody-carrying gelatin beads may be the first step toward the development of artificial CTL and can be applied, for example, to artificial dendritic and stroma cells for the development of novel biotherapeutic approaches.

[Case of stage IVb gastric cancer in which favorable anti-tumor effect and good QOL were observed due to UFTP therapy after low-dose CDDP-tegafur therapy].

A patient with stage IVb advanced gastric cancer, who was Group 4 lymph node metastasis positive, underwent two postoperative courses of low-dose CDDP-tegafur therapy (800 mg/body/day of tegafur + 5 mg/body/5 administrations, 2 days of rest, of cisplatin). UFTP therapy (400 mg/body/day of UFT + 5 mg/body/twice weekly of cisplatin) was thereafter given on an outpatient basis. The patient has now been receiving this therapy for one year and six months. The anti-tumor effect has been maintained and the tumor has been reduced in size by 89% without any adverse reactions. A good QOL has been observed. The present therapy can be performed safely at home and appears to be a favorable treatment from the viewpoint of QOL.

Development of a gelatin particle agglutination reagent for measles antibody assay.

A new agglutination test that utilizes gelatin particles as the carrier of measles antigen was developed and used to evaluate immune status to measles virus infection. The particle agglutination (PA) reagent reacted with monoclonal antibodies against two major proteins of measles virus, the hemagglutinin (H) and fusion (F) proteins. Children were followed individually for ten years for measles antibody. Results showed that the PA test was as sensitive and specific as the plaque neutralization test. The procedure is simple and rapid. No prior treatment of specimens is needed, and the test is completed in a single reaction. The PA test therefore can be used for diagnoses and epidemiologic surveys of measles virus infection.

Development and usefulness of the gelatin-particle-agglutination test for titration of antibodies against diphtheria, pertussis and tetanus toxins.

The gelatin-particle-agglutination (PA) test for titrating antibodies against diphtheria, pertussis and tetanus toxins was developed and used for assaying 65 sera from healthy children to assess the antitoxin acquisition in relation to the administration of adsorbed diphtheria-purified pertussis-tetanus (DPT) combined vaccine. The antitoxin titers obtained by the PA test and the conventional methods were correlated well; the correlation coefficient of the diphtheria antitoxin titers between the PA test and the cell culture method was 0.908, that of the tetanus antitoxin titers between the PA test and the passive hemagglutination test 0.968, and that of anti-pertussis toxin titers between the PA test and polystyrene-ball ELISA 0.885. The PA test was shown to be useful in both developed and developing countries, since it is simple to perform, sensitive and specific, and the three antitoxins can be titrated by the same procedure.

A novel magnetic particle agglutination in microtiter plates for rapid detection of human T-lymphotropic virus type I antibody.

We have developed a novel magnetic particle agglutination (MPA) method for rapid detection of antibody to human T-lymphotropic virus type I (HTLV-I). This method is suitable for screening determination. The assay uses purified viral antigen coated on magnetic particles, which are novel artificial particles made of gelatin, arabic gum, and ferrite. MPA uses a special magnet and inclined plate holder. In MPA, the specimen is incubated with magnetic particles in the well; magnetic particles are then magnetically attracted to the bottom of the well. Then the plate is inclined and read by the naked eye. In a positive specimen, agglutination particles stay at the bottom of the well. On the other hand, non-agglutination particles with negative sample run down the side of the well. The total assay time of MPA was estimated at 8 min/microtiter-plate. The results obtained by correlating MPA for the detection of HTLV-I antibody with other method emphasized the precision of MPA.

Improvement of simultaneous detection of antibodies to Gag and envelope antigens of human T-lymphotropic virus type I by western immunoblot assay.

To determine seropositivity for human T-lymphotropic virus type I (HTLV-I), we attempted to improve the detection system that uses antibody to HTLV-I Env in Western immunoblotting (WB) by adding an envelope glycoprotein (gp46) purified from the culture fluid of HTLV-I-producing cells by immunoaffinity chromatography and gel chromatography. In this WB, 177 of 179 serum samples showing seropositivity in an indirect immunofluorescence assay showed positive reactions to the gp46 envelope antigen as well as to p19, p24, and p53 Gag antigens. The remaining two samples showed negative reactions to p24. False-positive results were not found for 533 indirect immunofluorescence assay-negative serum samples, although one band to p19 or p24 was observed in 46 of the 533 samples. These 46 samples did not react to p53 and gp46, suggesting that these samples belonged to the indeterminate group in accordance with the criteria proposed by the World Health Organization. Therefore, this improved WB can be used for the confirmation of seropositivity.

Improvement of gelatin particle agglutination test for detection of anti-HTLV-I antibody.

Partial modifications of antigen components were made to improve the gelatin particle agglutination (PA) test for the detection of antibodies against human T cell leukemia virus type-I. Envelope glycoproteins prepared by lentil lectin affinity chromatography were further added to the purified viral antigens to be coated on the gelatin particles. Comparative studies with a conventional PA test kit (Serodia ATLA) and indirect immunofluorescence assay showed that the specificity and sensitivity of the new PA test were increased and that abnormal agglutination such as the prozone phenomenon was abolished by this improvement.

[Clinical observations with cefteram pivoxil granules in field of pediatrics].

A new oral cephem antibiotic, cefteram pivoxil (CFTM-PI, T-2588), was studied for clinical efficacy in the field of pediatrics. CFTM-PI was given orally to 23 patients with the following acute bacterial infections: 6 cases of acute tonsillitis, 8 of acute bronchitis, 2 of scarlet fever, 4 of bronchopneumonia, 1 of acute otitis media with sinusitis and 2 of urinary tract infections. Clinical results were "excellent" in 8, "good" in 14, "poor" in 1: the efficacy rate was 95.7%. As an adverse reaction, diarrhea was observed in 1 patient. From the above clinical results, it appears that CFTM-PI is a useful antibiotic for the treatment of pediatric patients with various bacterial infections.

Patterns of migration indicate sexual transmission of HTLV-I infection in non-pregnant women in Papua New Guinea.

The prevalence of infection with human T-cell leukaemia virus (HTLV-I) was studied in Madang Province on the north coast of Papua New Guinea. Serum specimens collected from non-pregnant women in 17 villages were tested for anti-HTLV-I by gelatin particle agglutination screening and confirmed by immunofluorescence and Western blotting. Overall, 13.9% of subjects were antibody-positive, with the prevalence of antibodies varying from less than 10% to 30% in villages situated less than 10 km apart. Two groups of migrant women were identified, and in both a parity-related increase in antibody prevalence which occurred only after marriage suggested that the predominant mode of transmission in migrant women was sexual. There was no parity-associated increase in anti-HTLV-I in indigenous women, and in contrast to migrant women, nulliparous indigenous women had a high prevalence of antibody (16.8% vs. 0%; p = 0.005). Vertical transmission cannot be excluded in indigenous women. No correlation was detected between the prevalence of anti-HTLV-I and a variety of indices of malarial infection.

Alteration in activities of Na,K, ATPase, sugar transport, and insulin receptors in erythrocytes from hyperthyroid patients.

To investigate the selectivity of the reduction in Na,K, ATPase activity in erythrocytes from patients with hyperthyroidism, we have assessed cytochalasin B-sensitive galactose uptake and insulin receptors as well as Na,K, ATPase-mediated rubidium uptake in erythrocytes from 11 patients with hyperthyroidism, 5 patients after treatment and 11 normal controls. There was a significant reduction in the Vmax values for rubidium uptake and the number of insulin receptors (23% and 20% below control, respectively), whereas there was a significant increase in the Vmax and Km values for galactose uptake (49% and 30% above control) and also in the average affinity of insulin receptors. The alterations both in rubidium and galactose transport activities were reversible with effective treatment of hyperthyroidism. The magnitude of alterations in the Vmax for rubidium uptake correlated inversely (r = -0.537 P less than 0.01, n = 16) and the Vmax and Km for galactose uptake correlated positively (r = 0.597 P less than 0.02 and r = 0.553 P less than 0.05, respectively) with serum T4 level. No correlation was found between the number of insulin receptors and serum IRI or T4 levels. These results suggest that the reduction in Na,K, ATPase activity observed in erythrocytes from hyperthyroid patients is not selective to this enzyme, but rather a reflection of widespread alterations of erythrocyte cell-surface proteins.

A new agglutination test for serum antibodies to adult T-cell leukemia virus.

A new gelatin particle agglutination test was developed for assay of natural antibodies to adult T-cell leukemia virus (ATLV/HTLV-I). Partially purified viral antigen from culture fluid of a virus-producer cell line was coated on artificial gelatin particle carriers. A high correlation was observed between the titers of antibodies determined by the agglutination test and by indirect immunofluorescence assay. The agglutination test is simple, sensitive and specific, and should be useful for mass screening of human sera for viral antibodies.

[A case of extramedullary plasmacytoma of the stomach].

A 51-year-old man with extramedullary plasmacytoma of the stomach is presented. He complained of epigastralgia. Barium meal revealed 2 submucosal tumors in the gastric body. Laboratory investigations showed neither hypergammaglobulinemia nor proteinuria. Bone X-ray examinations were not remarkable. There was tumor invasion to the pancreas and metastasis to the perigastric lymph nodes. Proximal gastrectomy was performed, and the patient died 5.5 years after the operation. Histologically, the proliferation of atypical plasma cells with eccentric nuclei was seen. Those cells invaded all layers of the stomach. The tumor cells were pyroninophilic and contained Russel bodies. Immunohistochemical study using the immunoperoxidase technique demonstrated IgG and lambda-light chains in the tumor cells.

[Changes in responsivity to TRH test and T3-suppression test after surgical treatment of hyperthyroidism (author's transl)].

TRH test and T3 suppression test were performed on patients with Graves' disease who underwent subtotal thyroidectomy after treatment with antithyroid drugs for 2.5 approximately 5 months. On 43 or the patients, TRH test was performed before, 1 week after and 1 approximately 2 months after surgery and T3 suppression test was also performed in 1 approximately 2 months post-operative period. For other 3 groups of the patients TRH test was performed at 2 approximatley 6 months, 6 approximately 12 months, 12 approximately 24 months and 24 approximately 41 months after surgery, and T3 suppression test was also performed just after each TRH test. As to TRH test, the response was defined as positive when basal TSH value was less than 2.0 muU/ml and peak value was more than 6.2 muU/ml or the difference between basal TSH and peak TSH value was over 5 muU/ml following TRH (500 mug) injection. T3 suppression test was perfomed by measuring the 24-hr thyroidal uptake of radioiodine after daily administration of 75 mug of T3 for 8 days. The response was defined as positive when the value for 24-hr uptake after T3 administration was less than half of the control value. The results were as follows; (1) In 11 of 43 patients, response to TRH test already changed to positive 1 week after operation and in 21 of 43 patients TRH test changes to positive 1 approximately 2 months after operation. (2) In general, response to TRH test changed to positive earlier than response to T3 suppression test. (3) T3 suppression test in 1 approximately 2 months after operation was useful to evaluate prognosis. (4) A half of the positive respondents to TRH test showed exaggerated response. (5) Basal TSH value of positive respondents to TRH test was 9.27 +/- 1.81 muU/ml (mean +/- SE) which exceeded the normal range. (6) Some patients showed negative response to conventional T3 suppression test despite of their high basal TSH value. This might be due to the insufficient dose of T3 to suppress TSH. (7) Concerning patients whose serum T3-RU, T4, T3 and TSH were within normal limit after subtotal thyroidectomy, 80% of them showed correspondance in the results of TRH test and T3 suppression test.