Production of murine hybrid-hybridomas secreting bispecific monoclonal antibodies for use in urease-based immunoassays

To produce bispecific antibodies (BiAbs) for enzyme immunoassay (EIA) to replace antibody-enzyme conjugates, we developed a panel of 8-azaguanine/ouabain-resistant anti-urease variant hybridoma cell lines for use in hybridoma-hybridoma fusions. These variants represent mouse immunoglobulin subclasses IgM, IgG1, IgG2a, and IgG2b and have growth rates equal to those of the parental hybridomas. We fused an anti-urease-secreting variant hybridoma with an anti-human choriogonadotropin (hCG)-secreting hybridoma (both of IgG1 subclass) and selected the desired product with growth media containing hypoxanthine-aminopterin-thymidine (HAT) and ouabain. Over 95% of the resulting hybrids secreted anti-urease, and 60% of these secreted anti-hCG. The bispecific nature of secreted antibodies was demonstrated in a simultaneous EIA where BiAbs, hCG, and urease (EC 3.5.1.5) were incubated together in anti-hCG-coated microwells. As little as 25 int. units of hCG per liter could be reliably detected, which is equivalent to that for antibody-enzyme conjugates in EIA.     

Effect of a histidine-excess diet on a tetrahydrofolylpolyglutamate pattern in rat liver

The effect of a histidine-excess diet on the hepatic folylpolyglutamate pattern in rat was studied. Rats were fed ad libitum 9.7% casein basal diets with 0.6% methionine (controls) or the basal diets with 3.5% histidine. The average daily weight gain and the food intake in histidine-supplemented rats (His-rats) did not significantly differ from controls. The liver weight in His-rats, however, was 50% higher than controls. Hepatic methyltetrahydropteroylpentaglutamate (CH3-H4PteGlu5), and tetrahydropteroylmonoglutamate concentrations in His-rats was 5.7- and 2-fold higher than controls, respectively. The tetrahydropteroylpentaglutamate (H4PteGlu5) concentration in the His-rats was 74% lower than controls. Considering the homeostasis of folate cofactors in tissues, these results suggest that the hepatic regeneration systems for H4PteGlu5 in His-rats might be repressed and an apparent methylfolate trap might be attained rather on a pteroylpentaglutamate level than a monoglutamate level, and that the activity for catabolizing the excess histidine might exceed the regenerating activity for folate cofactors.     

Angiotensin I-converting enzyme inhibitor improves reactive hyperemia in elderly hypertensives with arteriosclerosis obliterans.

Endothelial function in elderly hypertensive patients with arteriosclerosis obliterans has not been evaluated. We examined whether antihypertensive drugs improve vasodilatory response to reactive hyperemia of the limbs in elderly hypertensive patients (83 +/- 8 [SD] years) without (n=46, 0.9 < or = ankle-brachial pressure index < or = 1.4) and with (n=24) arteriosclerosis obliterans (ankle-brachial pressure index < 0.2). Patients were randomized for treatment with monotherapy of either temocapril (14 with and 26 without arteriosclerosis obliterans) or amlodipine (10 with and 20 without arteriosclerosis obliterans) for 6 months. Blood flows of the forearms and legs were measured by strain-gauge plethysmography. The vasodilatory response to the release of compression of the forearms and thighs at 200 mmHg or 20 mmHg more than systolic blood pressure for 5 min and to sublingual administration of nitroglycerin (0.3 mg) was assessed. The maximum reactive hyperemic flow in 35 legs with arteriosclerosis obliterans was significantly (p < 0.001) decreased compared to the value in legs in the control hypertensive subjects. Moreover, maximum reactive hyperemic flow in the forearms of patients with arteriosclerosis obliterans was significantly (p = 0.002) decreased compared to that in the control subjects. Blood pressure was similarly decreased by treatment with temocapril or amlodipine. Response to nitroglycerin (0.3 mg) was not changed by either drug. Treatment with temocapril significantly improved maximum reactive hyperemic flow of not only the legs and forearms in control hypertensives but also the legs and forearms in patients with arteriosclerosis obliterans, and attenuated the worsening of activity of daily living in these patients, although treatment with amlodipine did not. These results suggest that the angiotensin-converting enzyme inhibitor temocapril has a beneficial effect on endothelial function in elderly patients with arteriosclerosis obliterans.     

In vitro reactivity and in vivo biodistribution of the monoclonal antibody A7 using human gastric carcinoma cell lines.

The monoclonal antibody (MAb) A7 has been used to treat patients with colorectal or pancreatic carcinoma with encouraging results. We therefore determined if MAb A7 would also react with gastric carcinoma cell lines. MAb A7 reacted with seven of eight gastric carcinoma cell lines tested. The intensity of the reaction, measured by flow cytometry, was equal to that of WiDr (colon) and HPC-YS (pancreas) cell lines. In nude mice bearing xenografts of the MAb A7-reactive gastric cancer line MKN45, the percentage injected dose of MAb A7 per g of tumour tissue on day 7 was 9.79; this value was 77% of that on day 1. The in vivo tumour-to-blood ratio of MAb A7 was 2.77 on day 7. Therefore, MAb A7 has long-term retention at binding sites as well as a high probability, high intensity and high specificity of reactivity against gastric cancer, which make it an ideal drug carrier for immunotargeted chemotherapy and immunodiagnosis.     

Increase of striatal dopamine turnover by stress in MPTP-treated mice

The characteristics of motor function and brain dopamine (DA) metabolism in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice after immersion immobilization stress were investigated. There was no significant difference in locomotor activities between MPTP-treated and saline-treated mice, but locomotor activities of MPTP-treated mice after stress decreased more remarkably than those of saline-treated mice. Immediately after stress, striatal DA concentrations of MPTP-treated mice were significantly lower than those of saline-treated mice. Striatal DA levels improved when 24 h passed after stress. The striatal and cortical (DOPAC + HVA)/DA ratios of MPTP-and stress-treated mice was significantly higher than that of saline-and stress-treated mice. It is due to the decreased DA level and the enhancement of DA turnover that MPTP-treated mice became remarkably akinetic after stress, and that L-DOPA therapy is not effective when the symptoms in patients with Parkinson's disease worsen due to stress.     

Reconstructive surgery for supravalvular aortic stenosis after arterial switch operation for simple transposition in the early neonate

Recently, many papers have reported the development of severe pulmonary stenosis after an arterial switch operation (ASO) for the transposition of the great arteries (TGA) with intact ventricular septum (IVS) in the early neonate. However, supravalvular aortic stenosis after the same procedure is very uncommon. We experienced supravalvular aortic stenosis which developed gradually after successfully performing the Lecompte maneuver for TGA with IVS in a 6-day-old baby. We underwent Doty's extended aortoplasty in his 11th-month. We approached the aorta through the transection of the pulmonary trunk which was located just before the aorta. At the stenotic parts of the aorta, a discrete projection of cicatricial tissue was observed mainly at the posterior wall. The projection of the cicatricial tissue seemed to be induced as a consequence of the contraction of the aortic wall where a continuous suture was tied too tightly with non-absorbable suture materials. To prevent this complication after the Lecompte maneuver for TGA with IVS in the neonate, it is important to take care not to tie the suture too tightly and to use absorbable suture materials for construction of the aorta. There are the recommendations which we observed would prevent the post-operative complication of supravalvular aortic stenosis. As a final note, in repairing the supravalvular aortic stenosis, an approach through the transection of the pulmonary trunk was very useful.     

Experimental chronic dermatophytosis in human skin grafted to nude mouse: inoculation of Trichophytons and histopathological evaluation

Two strains of T. rubrum and one strain of T. mentagrophytes were inoculated into human skin grafted onto BALB/c nude mice by the needle puncture method. Infection was established in 1 of the 10 animals inoculated with fluffy colony type T. rubrum, 2 of the 10 animals inoculated with powdery colony type T. rubrum, and 7 of the 10 animals inoculated with granular colony type T. mentagrophytes, suggesting that the skin grafts are infectible by anthropophilic and zoophilic strains of dermatophytes. T. rubrum infection continued for a maximum of 9 weeks and T. mentagrophytes infection for more than 11 weeks. In the animals inoculated with T. mentagrophytes, fungal elements were localized in the stratum corneum of the human skin grafts. In the acute stage, microabscesses consisting of neutrophils were observed under the stratum corneum in contact with fungal elements; in the chronic stage, epidermal thickening and infiltration, mainly consisting of histiocytes and a smaller number of lymphocytes, was noted in the upper and middle dermis. Ultrastructural findings from the parasites were similar to those of dermatophytosis in man. This experimental system should be useful as a model of chronic dermatophyte infection in the human skin.