Vascular immunotargeting to endothelial surface in a specific macrodomain in alveolar capillaries.

A novel 85 kD glycoprotein (gp85) is a marker of the avesicular zone, a thin part of pulmonary endothelial cells separating alveolar and vascular compartments and lacking vesicles. This report presents the first evaluation whether mAb 30B3, a monoclonal antibody to gp85, can be used for targeting of drugs to the surface of lung endothelium. 125I-mAb 30B3 accumulated in isolated perfused lungs (IPL) (22.8 +/- 1.1 versus 0.5 +/- 0.1 %ID/g for 125I-IgG) and accumulated preferentially in the lungs after intravenous or intraarterial injection (10.9 +/- 0.7 and 11.0 +/- 1.5 versus 0.9 +/- 0.2 %ID/g for 125I-IgG). 125I-mAb 30B3 uptake in IPL was rapid (T1/2 15 min), saturable (Bmax appr. 10(5) molecules/cell), specific (inhibited by nonlabeled mAb 30B3) and temperature independent (26.3 +/- 2.1 versus 22.8 +/- 1.1 %ID/g at 6 degrees C versus 37 degrees C). Biotinylated mAb 30B3 permitted subsequent accumulation of perfused avidin derivative in IPL. Because these data indicated that mAb 30B3 binds to an accessible, poorly internalizable antigen in the lung, we conjugated mAb 30B3 with a plasminogen activator, 125I-tPA. After intravenous injection in rats, lung-to-blood ratio was 8.4 +/- 0.9 for mAb 30B3/125I-tPA versus 0.4 +/- 0.1 for IgG/125I-tPA, indicating that mAb 30B3 may deliver drugs, which was supposed to exert therapeutic action in the vascular lumen (e.g., antithrombotic proteins), to the surface of pulmonary endothelium.

Lung uptake of antibodies to endothelial antigens: key determinants of vascular immunotargeting.

Vascular immunotargeting is a mean for a site-selective delivery of drugs and genes to endothelium. In this study, we compared recognition of pulmonary and systemic vessels in rats by candidate carrier monoclonal antibodies (MAbs) to endothelial antigens platelet endothelial cell adhesion molecule (PECAM)-1 (CD31), intercellular adhesion molecule (ICAM)-1 (CD54), Thy-1.1 (CD90.1), angiotensin-converting enzyme (ACE; CD143), and OX-43. Tissue immunostaining showed that endothelial cells were Thy-1.1 positive in capillaries but negative in large vessels. In the lung, anti-ACE MAb provided a positive staining in 100% capillaries vs. 5-20% capillaries in other organs. Other MAbs did not discriminate between pulmonary and systemic vessels. We determined tissue uptake after infusion of 1 microg of (125)I-labeled MAbs in isolated perfused lungs (IPL) or intravenously in intact rats. Uptake in IPL attained 46% of the injected dose (ID) of anti-Thy-1.1 and 20-25% ID of anti-ACE, anti-ICAM-1, and anti-OX-43 (vs. 0.5% ID of control IgG). However, after systemic injection at this dose, only anti-ACE MAb 9B9 displayed selective pulmonary uptake (16 vs. 1% ID/g in other organs). Anti-OX-43 displayed low pulmonary (0.5% ID/g) but significant splenic and cardiac uptake (7 and 2% ID/g). Anti-Thy-1.1 and anti-ICAM-1 displayed moderate pulmonary (4 and 6% ID/g, respectively) and high splenic and hepatic uptake (e.g., 18% ID/g of anti-Thy-1.1 in spleen). The lung-to-blood ratio was 5, 10, and 15 for anti-Thy-1.1, anti-ACE, and anti-ICAM-1, respectively. PECAM antibodies displayed low pulmonary uptake in perfusion (2% ID) and in vivo (3-4% ID/g). However, conjugation with streptavidin (SA) markedly augmented pulmonary uptake of anti-PECAM in perfusion (10-54% ID, depending on an antibody clone) and in vivo (up to 15% ID/g). Therefore, ACE-, Thy-1.1-, ICAM-1-, and SA-conjugated PECAM MAbs are candidate carriers for pulmonary targeting. ACE MAb offers a high selectivity of pulmonary targeting in vivo, likely because of a high content of ACE-positive capillaries in the lungs.

Immunotargeting of glucose oxidase to endothelium in vivo causes oxidative vascular injury in the lungs.

Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti-PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H(2)O(2) generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H(2)O(2,) and caused endothelial injury manifested by a fourfold elevation of angiotensin-converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 +/- 9 vs. 2.4 +/- 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF(2alpha)-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo.

Streptavidin facilitates internalization and pulmonary targeting of an anti-endothelial cell antibody (platelet-endothelial cell adhesion molecule 1): a strategy for vascular immunotargeting of drugs.

Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.

Skin flap closure by dermal laser soldering: a wound healing model for sutureless hypospadias repair.

OBJECTIVES: Laser tissue soldering (LTS) with the diode laser and human albumin-hyaluronate-indocyanine green solder is a safe and effective method of providing an immediate leak-free closure during hypospadias repair. In this report, we compare the physiology, histology, and immunohistochemistry of wound healing following LTS and suturing in a rat skin flap model. METHODS: A 4 x 5-cm skin flap was raised and bisected (4 cm) on the dorsum of 48 Sprague-Dawley rats. The central wound was either closed from a dermal approach by suturing or LTS or left open, and studied at 0, 3, 5, 7, 10, 14, and 21 days postoperatively. An intraoperative comparison was made between suturing and LTS with respect to operative time. Postoperatively, flaps were excised for tensiometric analysis, and sections were stained with hematoxylin-eosin to define wound architecture. Resting skin temperature, laser exposed temperature without solder, and maximum temperature with solder (one drop) were measured at the level of the deep dermis, superficial striated muscle layer, and within the solder. Mean peak temperatures were recorded during a 1-minute laser activation time. RESULTS: Mean continuous suturing time (4.9 +/- 1.1 minutes) was significantly (P < 0.001) faster than either LTS (7.7 +/- 0.77 minutes) or discontinuous suturing (8.2 +/- 0.62 minutes). Two seromas (sutured) and two instances of partial wound dehiscence (1 sutured, 1 LTS) were noted. Tensile strength was increased significantly (P < 0.001) for up to 5 days in the LTS group, but was equal to suturing at 7 and 10 days. Immediate tensile strength after LTS was equivalent to a 7-day healed wound. At 14 days, wounds initially left open and those closed by LTS were stronger than sutured wounds (P < 0.05). There was no evidence of thermal injury or foreign body reaction in the LTS group. Solder was incorporated within the dermis in all wounds at 21 days. Laser activation of solder resulted in significant increases in temperature at all three tissue levels: 65.0 +/- 5.2 and 69.9 +/- 6.8 degrees C in the deep and superficial skin (no significant difference between the two), and 101 +/- 15.6 degrees C within the solder (P < 0.001 versus superficial and deep skin). CONCLUSIONS: Our results indicate that sutureless dermal LTS of skin flaps provides increased tensile strength for up to 7 days, with relatively greater tensile strength provided within the first 3 days. Our laser technique does not appear to alter the normal wound healing process. Rather, solder-tissue interaction initially, and extracellular matrix infiltration of solder later, provide the basis for improved wound strength. For hypospadias repair using skin flaps, these wound attributes may permit sutureless surgery.

Treatment of hepatic metastases with a combination of hepatic artery infusion chemotherapy and external radiotherapy.

Systemic chemotherapy has given good results when compared with that of patients who received no chemotherapy. When resection was performed, the results are slightly better. The group of patients who underwent combined therapy, using infusion chemotherapy, radiation and systemic chemotherapy, had the best results. It appears that infusion chemotherapy and radiotherapy can improve the palliation available for patients with metastases in the liver from carcinoma of the colon and rectum and warrants further investigation.

Electroosmotic characteristics of canine aorta and vena cava wall.

Experiments in which canine aorta and vena cava walls are subjected to electroosmosis in an open system at constant pressure are described. Electroosmosis reveals that the blood vessel walls studied have a negative zeta potential. The calculated zeta potentials are different for aorta and vena cava, -9.0 +/- 5.0 mv compared with -4.7 +/- 1.2 mv, respectively, and again of different magnitude with different bathing solutions. The calculated membrane pore charge per centimeter of effective pore surface in statcoulombs is approximately 6.2 x 10(3) for aorta compared with 3.5 x 10(3) for vena cava. The implications of the negative electroosmotic zeta potential in terms of the surrounding electric double layer, ion transport, and thrombosis are briefly discussed.