Renal Effects of Multiple Infusion of Pyridoxalated-Hemoglobin-Polyoxyethylene Conjugate (PHP) Solution in Dogs

Abstract: Pyridoxalated-hemoglobin-polyoxyethylene conjugate (PHP), which is made from out-dated human red blood cells by two major chemical modifications, namely pyridoxalation and conjugation with polyoxyethylene (POE), is currently under development as a physiological oxygen carrier. This study assessed the effects of PHP-88 solution, which contains 8% (wt/vol) each of hemoglobin (Hb) and maltose, on renal function when it was infused 3 times every other day into the intact circulation of 8 dogs (5 dogs for the PHP group and 3 for the control group; 20 ml/kg for the first infusion, and 10 ml/kg each for the second and third infusions, at the rate of 2.5 ml/h/kg). Serial determinations of glomerular filtration rate (GFR) and renal plasma flow (RPF) were carried out pre- and postinfusion for up to 3 months along with measurements of blood and urine analyses, urine output rate, fractional excretion of sodium (FES), and free water clearance (C_H2O). The results showed that plasma colloid osmotic pressure (COP) elevated an average of 3.3 mm Hg (p = 0.0085), and GFR and RPF tended to increase by 13% (NS) and 38% (NS), respectively, immediately after the third infusion with PHP solution. Urine output rate increased during and after the infusion, and FES and C_H2O also increased for 24 h after the infusion in both groups. Blood urea nitrogen, serum creatinine, and serum Na⁺ concentrations were not affected greatly by the infusions, but hematocrit was decreased by 8% in the PHP group, indicating approximately a 42% expansion of plasma volume. These changes were observed to return to their preinfusion levels by 1 week postinfusion. Renal histology of the PHP group obtained at 2 weeks postinfusion revealed vacuole formation in the proximal tubules which was not associated with any pathologic changes indicative of cell death or regeneration. In 4 out of 5 dogs at 3 months postinfusion (necropsy), the vacuoles were not present. Though urinary N-acetyl β-glucosaminidase (NAG) activity had significantly increased after infusion, it returned to the preinfusion level by 1 month postinfusion. No detrimental effect of vacuoles on the assessed renal tubular functions was confirmed in the present study. The result demonstrated that multiple infusions of PHP solutions were well tolerated in normal dogs, and the observed effects were conceived predominantly attributable to the physiological response of the kidneys to an oncotic load into the circulation, which produced plasma volume expansion.     

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.