Showing papers by "Rakesh K. Jain published in 1995"

Vascular Permeability in a Human Tumor Xenograft: Molecular Size Dependence and Cutoff Size

Abstract: Molecular size is one of the key determinants of transvascular transport of therapeutic agents in tumors. However, there are no data in the literature on the molecular size dependence of microvascular permeability in tumors. Therefore, we measured microvascular permeability to various macromolecules in the human colon adenocarcinoma LS174T transplanted in dorsal skin chambers in severe combined immunodeficient mice. These molecules were fluorescently labeled and injected i.v. into mice. The microvascular permeability was calculated from the fluorescence intensity measured by the intravital fluorescence microscopy technique. The value of permeability varied approximately 2-fold in the range of molecular weight from 25,000 to 160,000. These data indicate that tumor vessels are less permselective than normal vessels, presumably due to large pores in the vessel wall. The transport of macromolecules appears to be limited by diffusion through these pores. The cutoff size of the pores was estimated by observations of transvascular transport of sterically stabilized liposomes of 100-600 nm in diameter. We found that tumor vessels in our model were permeable to liposomes of up to 400 nm in diameter, suggesting that the cutoff size of the pores is between 400 and 600 nm in diameter.

Time-dependent Behavior of Interstitial Fluid Pressure in Solid Tumors: Implications for Drug Delivery

Abstract: Elevated interstitial fluid pressure (IFP) may constitute a significant physiological barrier to drug delivery in solid tumors. Strategies for overcoming this barrier have not been developed to date. To identify and characterize various mechanisms regulating IFP and to develop strategies for overcoming the IFP barrier, we modeled the tumor as a poroelastic solid. We used this model to simulate the effect of changes in microvascular pressure and tumor blood flow (TBF) on IFP. To test model predictions, the effects of changes in arterial pressure and TBF on IFP were measured using a tissue-isolated tumor preparation. IFP in the center of an isolated tumor was predicted to follow variation of the arterial pressure with a time delay of the order of magnitude of 10 s, and this delay was found to be 11 +/- 6 s experimentally. Following a cessation of TBF, the time constant of the drop in IFP was predicted to be of the order of 1000 s and was found to be 1500 +/- 900 s experimentally. The former time scale is characteristic of transcapillary fluid exchange, and the latter of percolation of fluid through the interstitial matrix. Relying on the good agreement between theoretical predictions and experimental data, we estimated the effect of blood pressure modulation on macromolecular uptake in solid tumors. Our results show that no appreciable increase of macromolecular uptake should occur either by an acute or by a chronic increase of blood pressure. On the other hand, higher uptake would result from periodic modulation of blood pressure. Therefore, the effectiveness of a vasoconstrictor such as angiotensin II to increase macromolecular delivery should be significantly enhanced by periodic rather than bolus or continuous administration of the vasoactive agent.

Scale-invariant behavior and vascular network formation in normal and tumor tissue.

Abstract: Tumor vascular networks look different from normal vascular networks, but the mechanisms underlying these differences are not known. By studying the scale-invariant behavior of normal and tumor vascular networks we show that vascular networks exhibit three classes of fractal behavior. Tumor networks display percolationlike scaling. Normal arteriovenous networks display diffusion-limited scaling, and normal capillary networks are compact structures. The mechanisms responsible for these differences are suggested using a growth model.

Biodistribution of monoclonal antibodies: scale-up from mouse to human using a physiologically based pharmacokinetic model.

Abstract: The efficacy of a novel diagnostic or therapeutic agent depends on its selective localization in a target tissue. Biodistribution studies are expensive and difficult to carry out in humans, but such data can be obtained easily in rodents. We have developed a physiologically based pharmacokinetic model for scaling up data from mice to humans, the first such model for genetically engineered macromolecules that bind to their targets in vivo, such as mAbs. The mathematical model uses physiological parameters including organ volumes, blood flow rates, and vascular permeabilities; the compartments (organs) are connected anatomically. This allows the use of scale-up techniques to predict antibody distribution in humans. The model was tested with data obtained in human patients for the biodistribution of a mAb against carcinoembryonic antigen. The model was further tested for a two-step protocol: bifunctional antibodies and radiolabeled hapten, which compared favorably with data in both mice and humans. The model was useful for optimization of treatment parameters, such as dose and time interval of injections, binding affinities, and choice of molecular carrier. This framework may be applicable to other genetically engineered molecules (e.g., growth factors, antisense oligonucleotides, and gene-carrying vectors).

Tumor Necrosis Factor α-induced Leukocyte Adhesion in Normal and Tumor Vessels: Effect of Tumor Type, Transplantation Site, and Host Strain

Abstract: Tumor necrosis factor α (TNF-α) can lead to tumor regression when injected locally or when used in an isolated limb perfusion, and it can enhance the tumoricidal effect of various therapies. TNF-α can also up-regulate adhesion molecules and, thus, facilitate the binding of leukocytes to normal vessels. The present study was designed to investigate the extent to which the host leukocytes roll and adhere to vessels of different tumors (MCaIV, a murine mammary adenocarcinoma; HGL21, a human malignant astrocytoma) at a given site or to the same tumor at different sites (dorsal skin and cranium), in different mouse strains [C3H and severe combined immunodeficient (SCID)], both with and without TNF-α-activation. There was no significant difference in hemodynamic parameters such as RBC velocity, diameter, or shear rate between PBS-treated control groups and corresponding TNF-α-treated groups. Under PBS control conditions, the leukocyte rolling count in MCaIV tumor vessels in the dorsal chamber in C3H and SCID mice and in the cranial window in C3H mice was significantly lower than that in normal vessels ( P < 0.05), but stable cell adhesion was similar between normal and tumor vessels. TNF-α led to an increase ( P < 0.05) in leukocyte-endothelial interaction in vessels in the following cases: normal tissue regardless of sites and strains, MCaIV tumor in the dorsal chamber in C3H and SCID mice, MCaIV tumor in the cranial window in C3H mice, and HGL21 tumor in the cranial window in SCID mice. However, the increase in rolling and adhesion in the MCaIV tumor in response to TNF-α was significantly lower than in the corresponding normal vessels ( P < 0.05) in the dorsal chamber in C3H and SCID mice and in the cranial window in C3H mice. The HGL21 tumor in the cranial window in SCID mice showed leukocyte rolling and adhesion comparable to that in normal pial vessels. These findings suggest that ( a ) in general, basal leukocyte rolling is lower in tumor vessels than in normal vessels; ( b ) leukocyte rolling and adhesion in tumors can be enhanced by TNF-α-mediated activation; and ( c ) the TNF-α response is dependent on tumor type, transplantation site, and host strain. These results have significant implications in the gene therapy of cancer using TNF-α-gene-transfected cancer cells or lymphocytes.

Characterization of a novel mucin sulphotransferase activity synthesizing sulphated O-glycan core 1,3-sulphate-Gal beta 1-3GalNAc alpha-R.

Abstract: A novel sulphotransferase (sulpho-T) activity from rat colonic mucosa was characterized using O-glycan core 1 substrate, Gal beta 1-3GalNAc alpha-benzyl. Derivatives of Gal beta 1-3GalNAc- were used to demonstrate that the 3- and 4-hydroxyl of Gal and the 2-acetamido group of the GalNAc residue of Gal beta 1-3GalNAc alpha-benzyl substrates were important for activity. Sulphated product using Gal beta 1-3GalNAc alpha-benzyl as substrate was analysed by ion spray mass spectrometry, methylation analysis, high-pH anion-exchange chromatography and beta-galactosidase digestion. The results suggested that sulphate was added to the 3-position of the Gal residue. The synthesis of core 2 from core 1 by UDP-GlcNAc: Gal beta 1-3GalNAc beta 6-GlcNAc-transferase was inhibited by sulphation of the Gal residue, indicating that GlcNAc beta 1-6 branching has to precede sulphation in the O-glycan core 1 processing pathway. These data demonstrate several novel pathways in the synthesis of sulphated mucin-type oligosaccharides.

Selectin ligands and tumor-associated carbohydrate structures: specificities of alpha 2,3-sialyltransferases in the assembly of 3'-sialyl-6-sialyl/sulfo Lewis a and x, 3'-sialyl-6'-sulfo Lewis x, and 3'-sialyl-6-sialyl/sulfo blood group T-hapten.

Abstract: The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 3(6-sulfo)GalNAc alpha- but was only 8% active with 6-sialo compound; it showed 11.6-fold greater activity with the copolymer of T-hapten. Further, we observed the alpha 2,3 sialylation of Gal beta 1,4GlcNAc beta- but not Gal beta 1,3GlcNAc beta- by HL60-ST, consistent with the occurrence of 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of sialylated or sulfated derivatives and acrylamide copolymers of Galβ1,3GalNAcα- and GalNAcα- to determine the specificities of blood group T- and Tn-specific lectins and the copolymers to measure anti-T and anti-Tn antibody levels in cancer patients

Abstract: Sialylated or sulfated derivatives and acrylamide copolymers of blood group T-(Galβ1,3GalNAcα-) and Tn-(GalNAcα) haptens were studied for their interaction with the lectins of peanut (PNA),Agaricus bisporus-(ABA),Helix pomatia-(HPA) andVicia villosa B4-(VVA), using asialo Cowper's gland mucin (ACGM), which contains both T and Tn epitopes, as the coating substrate in enzyme linked lectin assay. Both T and Tn copolymers (∼40 haptens) showed high affinity and strict specificity; although the T-copolymer at 0.05–0.07 µm concentration caused 50% inhibition of interaction of either PNA or ABA with ACGM, there was little inhibition of the HPA and VVA interactions at over 100 times that concentration. The Tn-copolymer at 0.02–0.05 µm inhibited HPA or VVA interaction with ACGM by 50% but gave virtually no inhibition of PNA and ABA binding. Sialyl, sulfate or methyl group substitution on C-6 of GalNAc of the T-haptene did not prevent interaction with PNA but almost abolished interaction with ABA. In contrast, sialyl or sulfate group on C-6 and sulfate on C-3 of Gal in Galβ1,3GalNAcα- inhibited almost completely the interaction of PNA with ACGM but had only a slight effect on the interaction of ABA; C-6 substitution with either sialic acid or sulfate on GalNAcα- almost abolished the interaction of both HPA and VVA with ACGM. Preliminary studies revealed a significant depression in the serum level of anti-T (two to three-fold decrease) and anti-Tn (∼ two-fold decrease) antibodies in breast cancer compared with normal control subjects when the acrylamide T- and Tn-copolymers were used as coating substrates in enzyme linked immunoassays.

Expression of blood group Lewis b determinant from Lewis a: association of this novel alpha (1,2)-L-fucosylating activity with the Lewis type alpha (1,3/4)-L-fucosyltransferase.

Abstract: Blood group H type 1 [Fuc alpha (1,2)Gal beta (1,3)GlcNAc beta-->] is known as the precursor structure of the blood group determinant, Lewis b [Fuc alpha (1,2)Gal beta (1,3)(Fuc alpha (1,4))GlcNAc beta-->]. Recently, a new biosynthetic route for Lewis b from Lewis a [Gal beta (1,3)(Fuc alpha (1,4))GlcNAc-->] was identified in human gastric carcinoma cells, colon carcinoma Colo 205, and ovarian tumor. The present study demonstrates the association of this new type of alpha (1,2)-L-fucosyltransferase (FT) activity with the Lewis-type alpha (1,3/4)-L-FT as follows: (i) the alpha (1,4)- and novel alpha (1,2)-FT activities of Colo 205 were much less inhibited than the alpha (1,3)-FT activity by N-ethylmaleimide [Ki(microM) = 714.0, 119.0, and 6.5 respectively]. (ii) The alpha (1,4)- and novel alpha (1,2)-FT activities emerged from a Sephacryl S-200 column in identical positions. (iii) A specific inhibitor (copolymer from 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-allyl and acrylamide) of alpha(1,4)-FT activity inhibited both alpha(1,4)- and alpha(1,2)-FT activities in Sephacryl S-200 column effluent to almost the same extent (approximately 80%); (iv) separation of the Lewis-type alpha(1,3/4)-FT from the plasma-type alpha(1,3)-FT by specific elution of the affinity column (bovine IgG glycopep-Sepharose) with lactose and further purification on a Sephacryl S-100 HR column showed that (a) the alpha(1,3)-FT activity was the inherent capacity of the Lewis-type FT (Colo 205 fraction L) since approximately 90% of both the alpha(1,4)- and alpha(1,3)-FT activities is inhibited by the copolymer, (b) the unique ability of catalyzing the alpha(1,2)-L-fucosylation of Gal in Lewis a structure and also the alpha(1,3)-L-fucosylation of Glc in lactose-based structure belonged to the Lewis type enzyme (Colo 205 fraction L), (c) a measurement of the [14C]fucosyl products arising from the two acceptors Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn and 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A1 (specific for alpha(1,2) and alpha(1,4), respectively) taken in the same incubation mixture showed mutual inhibition by the acceptors ([Km for the alpha(1,4)-specific acceptor, 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A], increased from 32 to 50 microM in the presence of 7.5 mM Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn, whereas Ki for the mutual inhibition of alpha(1,2)-FT activity by the former was 102 microM], and (d) the Lewis-type FT, in contrast to the plasma type FT, was highly effective in fucosylating complex glycopeptides. (iv) A cloned FT (FT III:Lewis type) and the Colo 205 Lewis-type FT (fraction L) showed similar activities toward various acceptors; the enzymatic product resulting from the action of cloned FT on Galbeta(1,3)(Fucalpha(1,4))GlcNAc-beta-O-Bn was identified by FAB mass spectrometry as the difucosyl compound. (v) An examination of six human cell lines indicated that the novel alpha(1,2)-FT activity associates with the alpha(1,4)-FT activity.

Kinetics of adhesion molecule expression and spatial organization using targeted sampling fluorometry.

Abstract: Cellular interactions with the vascular wall under flow conditions are controlled, in part, by the density of adhesion molecules on endothelial cells. The spatial arrangement and absolute levels of these molecules over the endothelium are therefore important determinants of cellular localization. Many biochemical and functional studies have characterized the interactions between leukocytes and endothelial monolayers, but no reliable method has been reported for quantifying the spatial expression of adhesion molecules on intact endothelial cell monolayers. We report the development of targeted sampling fluorometry (TSF), which uses standard immunostaining, fluorescence microscopy and digital image analysis techniques to analyze cell surface molecule expression on a cell-by-cell basis. This technique is performed on an intact monolayer and results in cellular intensity distributions that reflect spatial heterogeneity in adhesion molecule expression. We demonstrate the use of targeted sampling fluorometry in a study of the kinetics of tumor necrosis factor alpha-induced activation of human umbilical vein endothelial cell monolayers and show that the spatial patterns of adhesion molecule expression correlate with the locations of bound lymphocytes.

Quantitative analysis of angiogenesis and growth of bone: effect of indomethacin exposure in a combined in vitro-in vivo approach.

Abstract: Nonsteroidal anti-inflammatory agents have been used experimentally and clinically to suppress a variety of physiological events, including angiogenesis and formation of bone. The exact mechanisms by which indomethacin alters skeletal tissue generation are unknown, due in part to methodological limitations. By the use of an organ culture assay and an animal model using intravital microscopy in mice bearing dorsal skinfold chambers, the effect of indomethacin on growth and angiogenesis of neonatal femora was characterized over 16 days. In both assays, femora significantly elongated with time (P<0.05). The in vitro growth rate was more rapid than in vivo and dependent on the serum concentration, culture medium and age of mice. Although enthancing the serum content promoted cellular proliferation in organ culture, it dose-dependently suppressed femoral elongation, leading at 20% fetal calf serum to growth rates identical to those observed in vivo. Indomethacin supplementation (2 and 10 mg l−1) significantly accelerated longitudinal femoral growth in organ culture (P<0.05), whereas in vivo indomethacin (2 mg kg−1) did not modulate either angiogenesis or elongation of bone. Our in vitro data propose a central role of serum in the regulation of bone formation. Although indomethacin altered femoral gowth in vitro, our findings do not suggest that indomethacin suppresses angiogenesis or growth of bone in vivo. The complexity of physiological events in vivo may be obscuring a detectable effect.

Selectin ligands: synthesis of 3′-O-sialyl-6′-O-sulfo Lewis, NeuAcα2→3(6-O-SO3Na)Galβ1→3 (Fucα1→4) GlcNAcβ–OMe

Abstract: The stereoselective synthesis of 3′-O-sialyl-6′-O-sulfo-Le, a stereoisomer of the major capping group of GLYCAM-I is described.

Disseminated cutaneous herpes zoster: A clinical predictor of human immunodeficiency virus infection.

Abstract: A 45-year-old woman presented with thoracic (3,4,5) herpes zoster with cutaneous dissemination. She was found positive for human immunodeficiency virus infection. Mucocutaneous examination revealed presence of oral thrush and oral hairy leukoplakia. The patient possibly acquired the infection through blood transfusion.