Identification of tumor vascular antigens by monoclonal antibodies prepared from rat-tumor-derived endothelial cells

An industrial erythromycin production strain of Saccharopolyspora erythraea spp. was used to demonstrate that careful genetic engineering can significantly improve productivity. The chromosomally integrated Vitreoscilla hemoglobin gene (vhb) was shown to enhance the final titer of erythromycin by some 70% compared to the original S. erythraea spp. Overall, specific erythromycin yields were about 2.5 g of erythromycin/g of total protein for S. erythraea::vhb but <1 for the S. erythraea spp. The maximum rates of biosynthesis were 57.5 mg of erythromycin/(L/h) and 24.3 mg/(L/h) for the recombinant strain S. erythraea::vhb and S. erythraea spp., respectively. Overall space-time yield was 100% higher for the S. erythraea::vhb fermentation (1.1 g of erythromycin/(L/day)) than for the S. erythraea spp. fermentation (0. 56 g of erythromycin/(L/day)). The genetic stability of the recombinant strain was high, and no selective pressure was needed throughout the cultivations. Expression of functional Vitreoscilla hemoglobin throughout the cultivations was verified by CO difference spectrum assays.     

Monoclonal antibodies against vascular endothelial antigens expressed in normal human brain

A panel of monoclonal antibodies reactive with human-brain vessels was raised by immunizing BALB/c mice with homogenate of whole human brain, obtained from temporal lobectomies. Hybridoma supernates were screened by immunohistochemical methods on frozen sections of human brain, liver and spleen and 16 clones were isolated. The pattern of immunoreactivity varied with respect to the type of brain blood vessels predominantly labelled and to tissue specificity. Some antibodies cross-reacted with cow or squirrel monkey forebrain microvessels with an intensity equal to that shown by human brain. The immunoreactivity patterns reflected antigenic heterogeneity among different subsets of vascular endothelial cells in human brain.     

A radioimmunoassay for antibodies against surface membrane antigens using adhering cells

A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit 125I-anti-mouse-Fab. The bound radioactive material was eluted with glycine-HCI buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolytic or noncyctolytic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface.     

Occurrence and specificity of human natural and in vitro induced antibodies to Nocardia opaca antigens

Nocardia opaca, a Gram-positive bacterium, is a potent source of immunostimulatory substances. Screening of sera of adult human donors revealed that all sera tested contained antibodies reactive with isolated Nocardia fractions (Nocardia delipidated cell mitogen, NDCM; Nocardia lysozyme digest, NLD; Nocardia water-soluble mitogen, NWSM; and fraction B). The respective values of reciprocal titres for IgM and IgG were in the range of 100 to 12,800, and 10 to 320 for IgA antibody isotypes, when NLD or fraction B were used as antigens in enzyme-linked immunosorbent assay (ELISA) tests. The level of antibodies directed to NDCM, a potent polyclonal B cell activator, was found to be the lowest. In vitro spontaneous as well as NDCM-induced production of antibodies to NDCM by human peripheral blood lymphocytes involved mainly the IgM class. Western-blot analysis demonstrated that antibodies in normal human sera react with nocardial antigens of molecular mass approximately 60, 40, 20 and 15-10 kDa. The same antigens were also recognized by rabbit and mouse hyperimmune sera, also confirming the immundominancy of these nocardial antigens in other species. The presence of anti-nocardia antibodies in human sera and their production by both stimulated and non-stimulated lymphocytes points to the natural sensitization of humans either by ubiquitous no-cardial components or by cross-reactive bacterial or food antigens.     

Electrical coupling between smooth muscle cells and endothelial cells in pig coronary arteries

The control of smooth muscle cells by endothelial cells has been well established by the identification of vasoactive factors released by the endothelial cells. In contrast, the possibility that smooth muscle cells influence the endothelial cells has been considered rarely. Some results suggest possible electrical communication between the smooth muscle and the endothelial cells but proof is lacking. We therefore tested for electrotonic conduction of signals from smooth muscle cells to endothelial cells. The endothelium was removed from half of a strip of porcine coronary artery. In a partitioned chamber, rectangular hyperpolarization or depolarization was applied to the de-endothelialized region by field stimulation. The resulting membrane potential changes in the smooth muscle cells spread electrotonically along the media into the area with intact endothelium. We recorded from endothelial cells to determine whether this electrical signal spreads into endothelial cells. Hyperpolarization or depolarization initiated in smooth muscle cells was recorded consistently in endothelial cells. This demonstrates a functional electrotonic propagation from smooth muscle to endothelial cells.     

Distribution of blood-group-related carbohydrate antigens on oral endothelial cells

Recent studies indicate that carbohydrate structures are involved in various endothelial functions such as inflammatory processes, adhesion of metastatic cells to endothelium and endothelial differentiation. In this paper we report the endothelial expression of various blood-group-related carbohydrate structures in normal oral mucosa using 15 different monoclonal antibodies reacting with type 1, 2 or 3 carbohydrate chains. Twenty biopsies, including normal oral mucosa, from secretor individuals comprised nine blood group O, nine A, one B and one AB. Endothelial staining was compared with epithelial staining in the same biopsies. Five blood-group-related carbohydrate antigens were detected on endothelial cells. The H type 2 antigen, which is the precursor of A, B and AB antigens, has previously been believed to be a universal marker for endothelial cells. All blood group O individuals (n = 9) showed strong H antigen staining in the endothelium of most vessels. However, of blood group A, B and AB individuals (n = 11), four showed heterogenous H antigen staining. In addition, we found that six out of ten blood group A or AB individuals, who expressed A or A and B antigens on spinous squamous cells and glandular epithelium, showed either heterogenous or no staining for these structures on their endothelial cells. It is concluded that there are differences between the biosynthesis of blood-group-related carbohydrate antigens in oral endothelium and epithelium.     

Comparative study of the effects of clozapine and clothiapine in different preparations of guinea pig and rat isolated organs

1. A study has been made to know the effects of clozapine and clothiapine on the responses of rat isolated vas deferens to norepinephrine, dopamine and potassium, those of the rat isolated uterus to serotonin and potassium, and that of guinea pig isolated ileum to histamine. 2. Clozapine was a noncompetitive antagonist to norepinephrine, dopamine, serotonin and histamine; it inhibited potassium-induced contraction in isolated rat uterus and vas deferens. 3. Clothiapine was a competitive antagonist to serotonin and a noncompetitive antagonist to norepinephrine, dopamine and histamine; it inhibited potassium-induced contractions in isolated rat uterus and vas deferens.     

Preferential involvement of Go and Gz proteins in mediating rat natural killer cell lysis of allogeneic and tumor target cells

IL-2-activated NK cells from PVG rats potently lyse target cells expressing allo-MHC class I determinants. Here, we investigated the role that G proteins play in mediating this activity. Pretreatment of NK cells with pertussis toxin (PT) or cholera toxin (CT) inhibited NK cell killing of tumor (YAC-1 or P815), and allogeneic target cells. ADP ribosylation assay revealed that PT ADP ribosylates a 39-kDa G protein, whereas CT ADP ribosylates a 45 to 47-kDa G protein in PVG NK cell membranes. Membranes prepared from intoxicated NK cells with either PT or CT lost their ability to incorporate [32P]NAD. These membranes possess Gi, Go, Gs, and Gz as demonstrated by immunoblot analysis. However, Gq was not clearly detected by this method. IL-2-activated NK cells were permeabilized with streptolysin O. Permeabilized cells incorporated Abs to Gi, Go, Gz, Gs, and Gq as determined by flow cytometric analysis. When Abs to Go or Gz, but not to Gi, Gs, or Gq, were incorporated inside permeabilized NK cells, a significant reduction in the lysis of tumor or allo-MHC target cells was observed, suggesting that Go and Gz play important roles in transducing the signals necessary to lyse target cells. Our results show for the first time a role for G proteins in mediating NK cell killing of allo-MHC-encoded target cells, and provide evidence for Gz protein involvement in NK cell recognition of target cells. The effect of Gz is novel and has not been previously described in any other system or cell type.     

A dual role for endothelial cells in cytomegalovirus infection? A study of cytomegalovirus infection in a series of rat endothelial cell lines

Several clinical findings point to the involvement of microvascular endothelial cells in cytomegalovirus-related pathology. In this study the interactions of cytomegalovirus (CMV) with microvascular endothelial cells was investigated in an in vitro rat model. A series of rat endothelial cell lines, considered representative for the heterogeneity of heart microvascular endothelium in vivo, were infected with rat CMV (RCMV). The course of infection and production of infectious virus were examined using immunofluorescence staining and plaque titration assays, and was compared with infection of fully permissive rat fibroblasts. These endothelial cell lines displayed differences in susceptibility to CMV infection. Two endothelial cell lines (RHEC 50 and 191) were practically non-permissive, while four endothelial cell lines (RHEC 3, 10, 11 and 116) were partly permissive for CMV infection. In contrast to CMV infection in fibroblasts, only limited infection of the permissive endothelial cell lines was observed without spreading of CMV infection through the monolayer, although infectious virus was produced. Detachment of infected endothelial cells and recovery of the monolayer with time was observed. The detached endothelial cells were able to transmit CMV infection to fibroblast monolayers, but not to endothelial monolayers. Our in vitro results demonstrate differences in permissiveness for RCMV between the series of rat endothelial cell lines, which is suggestive for endothelial heterogeneity to CMV infection in vivo. Our findings indicate that endothelial cells are relatively resistant to CMV infection and that, upon infection, the endothelial monolayer may dispose of the virus via detachment of the infected cells. This points to a dual role for the endothelium in CMV infection in vivo: a barrier for CMV infection (by the endothelial monolayer) on the one hand and spreading of CMV infection (by detached infected cells) on the other hand.     

Activation of cultured vascular endothelial cells by antiphospholipid antibodies

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.     

Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice

This study sought to determine whether angiogenic blood vessels in disease models preferentially bind and internalize cationic liposomes injected intravenously. Angiogenesis was examined in pancreatic islet cell tumors of RIP-Tag2 transgenic mice and chronic airway inflammation in Mycoplasma pulmonis-infected C3H/HeNCr mice. For comparison, physiological angiogenesis was examined in normal mouse ovaries. We found that endothelial cells in all models avidly bound and internalized fluorescently labeled cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]/cholesterol or dimethyldioctadecyl ammonium bromide [DDAB]/cholesterol) or liposome-DNA complexes. Confocal microscopic measurements showed that angiogenic endothelial cells averaged 15-33-fold more uptake than corresponding normal endothelial cells. Cationic liposome-DNA complexes were also avidly taken up, but anionic, neutral, or sterically stabilized neutral liposomes were not. Electron microscopic analysis showed that 32% of gold-labeled liposomes associated with tumor endothelial cells were adherent to the luminal surface, 53% were internalized into endosomes and multivesicular bodies, and 15% were extravascular 20 min after injection. Our findings indicate that angiogenic endothelial cells in these models avidly bind and internalize cationic liposomes and liposome-DNA complexes but not other types of liposomes. This preferential uptake raises the possibility of using cationic liposomes to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors and sites of chronic inflammation.     

Nitric oxide production by pig endothelial cells in response to human-derived injury

BACKGROUND: Metamorphopsia is a visual illusion that distorts the size, shape, or inclination of objects. Reversal of vision metamorphopsia (RVM) is a rare transient form of metamorphopsia described as an upside-down, 180 degrees rotation of the visual field in the coronal plane. The pathophysiological characteristics of RVM remain unclear. DESIGN: Patients with RVM had a complete neurologic examination during or shortly after an episode of metamorphopsia, with particular emphasis on gaze disorders, visual fields, visually guided hand movements, and perceptual or cognitive deficits. Workup included imaging studies, visual field examinations, and brainstem auditory and visual evoked response. SETTING: Department of Neurology, Hadassah University Hospital, Hebrew University-Hadassah Medical School, Jerusalem, Israel. PATIENTS: Six consecutive patients were evaluated from 1991 to 1996. RESULTS: Five patients had parieto-occipital brain insult sparing the primary visual cortex, and 3 also had evidence of a concomitant brainstem or cerebellar syndrome. One patient had pure brainstem syndrome underlying the RVM. Three patients had complete RVM as well as oblique RVM of less than 180 degrees. CONCLUSIONS: These cases imply a possible anatomical localization of the central integrator of visual extrapersonal orientation. Our observations suggest that a separate central mechanism of visual orientation might exist in each cerebral hemisphere and that occipital and parietal lesions that spare the optic radiations may account for the oblique and complete RVM. We postulate that failure to perceive space in an allocentric coordinate frame, particularly in the coronal roll plane, is potentially the critical event underlying RVM.     

Comparative immunochemistry and ontogeny of two closely related coated pit proteins. The 280-kd target of teratogenic antibodies and the 330-kd target of nephritogenic antibodies

We have previously shown that monoclonal antibodies specific for a 280-kd protein (gp280) concentrated within the coated pits of renal and yolk sac brush border-induced fetal malformations, whereas antibodies specific for gp330, another coated pit protein with a similar distribution, had no deleterious effect on embryonic development. In this study, we show that gp280 and gp330 are closely related proteins, as indicated by: 1) similarities in peptide maps obtained after cyanogen bromide cleavage, 2) immunological cross-reactivity related to a minor contingent of antibodies that do not have teratogenic activity, and 3) asynchronous but related expressions during ontogenesis. During the early stages of development, the expression of the two glycoproteins was limited to (gp330) or predominant in (gp280) the clathrin-coated pits and intermicrovillar areas. In the pre-implantation embryo, gp330 was expressed by trophectodermal cells, which became negative in day-6 embryos trapped in endometrial infoldings. At this stage, gp280 and gp330 were both simultaneously detectable at the apical pole of the first entoblastic cells and remained expressed by the brush border of visceral yolk sac epithelial cells until the end of pregnancy. In addition, gp330 was expressed by amniotic cells and neurectodermal structures. During nephrogenesis, in contrast, the expression of gp280 and gp330 by the intermicrovillar areas of the proximal tubule cell was the result of a complex maturation process. gp280 and gp330 were diffusely distributed in S-shaped bodies in the presumptive areas of the glomerulus, proximal tubule, and distal tubule (gp330). During development of the nephron, the pattern of expression became progressively restricted to the proximal tubule and glomerulus (gp330), and selective localization in the intermicrovillar areas was only achieved in filtrating nephrons.     

Compatible organic osmolytes in rat liver sinusoidal endothelial cells

Vanadium has been found to be orally active in lowering plasma glucose levels; thus it provides a potential treatment for diabetes mellitus. Bis(maltolato)oxovanadium(IV) (BMOV) is a well-characterized organovanadium compound that has been shown in preliminary studies to have a potentially useful absorption profile. Tissue distributions of BMOV compared with those of vanadyl sulfate (VS) were studied in Wistar rats by using 48V as a tracer. In this study, the compounds were administered in carrier-added forms by either oral gavage or intraperitoneal injection. Data analyzed by a compartmental model, by using simulation, analysis, and modeling (i.e., SAAM II) software, showed a pattern of increased tissue uptake with use of 48V-BMOV compared with 48VS. The highest 48V concentrations at 24 h after gavage were in bone, followed by kidney and liver. Most ingested 48V was eliminated unabsorbed by fecal excretion. On average, 48V concentrations in bone, kidney, and liver 24 h after oral administration of 48V-BMOV were two to three times higher than those of 48VS, which is consistent with the increased glucose-lowering potency of BMOV in acute glucose lowering compared with VS.     

A comparative study of guinea pig and rat tracheal reactivity

Species differences in behavior and responses of guinea pig (GPT) and rat (RT) tracheal smooth muscle to electrical field stimulation (EFS) and chemicals were investigated under semi-isometric conditions. Indomethacin augmented and papaverine reduced the contractions elicited by EFS in both tissues. They relaxed the GPT and did not affect the tone decline in the RT. Experiments with different load, cartilage content, thread and rubber strips suggest the role of elastic elements in passive elongation of the RT. This was independent of muscarinic, adrenergic, histaminergic and serotoninergic receptors, nitric oxide, eicosanoids and metabolic processes. The second response on repeated administration of acetylcholine was augmented and that of serotonin attenuated in both GPT and RT. Upon repeated elevation of [K+]o the tissues responded in an opposite manner. Further data are provided on similarities (modulation of cholinergic transmission by prostaglandins, sensitization to acetylcholine and desensitization to serotonin) and differences (innervation, responses to repeated elevation of [K+]o, presence/absence of prostaglandin-regulated basal tone and spontaneous activity) in reactivity of GPT and RT.