Natural killer cells infiltrating colorectal cancer and MHC class I expression.

A majority of colorectal adenocarcinomas displays diminished MHC class I expression, making them particularly vulnerable for NK cell-mediated killing. Generally, these tumors also show a substantial inflammatory infiltrate. Most inflammatory cells, however, reside in the tumor stroma, where they do not have direct contact with tumor cells in the tumor epithelium. In this study, we investigated the correlation between colorectal tumor MHC class I aberrations and infiltration of NK cells. We studied 88 tumor specimens obtained from 88 colorectal cancer patients for locus-specific HLA aberrations and correlated these data to infiltration of CD4, CD8+ and CD56+ lymphocytes. The lymphocyte markers were individually combined with laminin as a second marker to facilitate quantification in the different tumor compartments, i.e. tumor epithelium and tumor stroma. Locus-specific partial or total HLA class I loss was detected in 72% of the tumors studied. Twenty-eight percent had no HLA loss at all. Mean overall intra-epithelial infiltration of CD56+ lymphocytes was 7 cells/mm(2) compared to 76 cells/mm(2) for CD8 and 19 cells/mm(2) for CD4+ lymphocytes. Locus-specific partial or total loss of tumor cell MHC class I expression was positively correlated with the intra-epithelial infiltration of CD8+ cells (P = 0.01), but not with CD4+ or CD56+ lymphocytes. Triple immunofluorescence staining showed that these cells were CD8 and granzyme-B positive T-lymphocytes. Our data showed that colorectal tumors are sparsely infiltrated by CD56+ cells compared to CD8+ T-cells and that loss of MHC is associated with T-cell infiltration instead of NK cell infiltration. Considering the fact that MHC loss is quite common in colorectal cancer and that, due to local absence of NK cells, it is unlikely that there has been selection for NK-escape variants, improvement of the intra-epithelial infiltration/migration of NK cells may be an important basis for the development of an effective adjuvant NK-based immunotherapy of colorectal cancer.

Tumor blood supply and tumor localization by adoptively transferred IL-2 activated natural killer cells.

The circulatory pattern of IL-2 activated natural killer (A-NK) cells was studied in C57BL/6 mice bearing 10 day-old pulmonary and subcutaneous (s.c.) metastases of the B16 melanoma in order to evaluate the roles of the concentration of A-NK cells in the blood and of tumor blood flow on accumulation of A-NK cells in tumors. Kinetic studies of the presence of A-NK cells in peripheral blood after adoptive transfer revealed that these cells rapidly disappear from the blood. Via intravital microscopy of animals with exposed lung tissue, we have shown that the vast majority of transferred A-NK cells become efficiently arrested within the lung microcirculation at their first encounter with this organ, thereby explaining the fast disappearance of the cells from the bloodstream. Despite the low number of A-NK cells circulating in the blood, systemically injected A-NK cells (20 million per mouse) localized significantly (70-80 million cells/g) into most pulmonary metastases within 8-16 hours. In contrast, very few A-NK cells (< 0.2 million cells/g) were found in the s.c. metastases. Based on measurements of tumor blood flow (showing a classic inverse relationship between tumor size and tumor blood flow) and the blood concentration of A-NK cells, we estimated the highest intratumoral density of A-NK cells that theoretically can be generated by A-NK cells transported to the tumor by way of the blood. In s.c. tumors, the observed density of A-NK cells was at all times lower (10-50 fold) than the estimated density, indicating that only a few percent of the A-NK cells arriving at these tumors become retained in them. In contrast, the observed density of A-NK cells in pulmonary metastases was at all times higher (2-3 fold) than the estimated density. This finding indicates that A-NK cells might not reach the pulmonary metastases solely by way of the blood stream. In conclusion, i.v. injected A-NK cells become immediately entrapped in the lungs and, consequently, circulate poorly. While lung metastases become significantly infiltrated by i.v. injected A-NK cells, metastases in organs down-stream from the lungs become poorly infiltrated. We hypothesize that only a part of the A-NK cells found in lung metastases 8-16 hours following injection reach these metastases by way of the blood-vascular system. They might also migrate into the metastases from the surrounding normal lung tissue.

Photosystem II in different parts of the thylakoid membrane: a functional comparison between different domains.

The electron transport properties of photosystem II (PSII) from five different domains of the thylakoid membrane were analyzed by flash-induced fluorescence kinetics. These domains are the entire grana, the grana core, the margins from the grana, the stroma lamellae, and the Y100 fraction (which represent more purified stroma lamellae). The two first fractions originate from appressed grana membranes and have PSII with a high proportion of O(2)-evolving centers (80-90%) and efficient electron transport on the acceptor side. About 30% of the granal PSII centers were found in the margin fraction. Two-thirds of those PSII centers evolve O(2), but the electron transfer on the acceptor side is slowed. PSII from the stroma lamellae was less active. The fraction containing the entire stroma has only 43% O(2)-evolving PSII centers and slow electron transfer on the acceptor side. In contrast, PSII centers of the Y100 fraction show no O(2) evolution and were unable to reduce Q(B). Flash-induced fluorescence decay measurements in the presence of DCMU give information about the integrity of the donor side of PSII. We were able to distinguish between PSII centers with a functional Mn cluster and without any Mn cluster, and PSII centers which undergo photoactivation and have a partially assembled Mn cluster. From this analysis, we propose the existence of a PSII activity gradient in the thylakoid membrane. The gradient is directed from the stroma lamellae, where the Mn cluster is absent or inactive, via the margins where photoactivation accelerates, to the grana core domain where PSII is fully photoactivated. The photoactivation process correlates to the PSII diffusion along the membrane and is initiated in the stroma lamellae while the final steps take place in the appressed regions of the grana core. The margin domain is seemingly very important in this process.

Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling.

Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from baker's yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzoyl dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na2SO4.

Beneficial effect of treatment with transdermal estradiol-17-beta on exercise-induced angina and ST segment depression in syndrome X.

We investigated the acute effect of transdermal estradiol-17-beta on exercise-induced ischemia in 15 postmenopausal women (mean age 58 +/- 6 years) with syndrome X (angina pectoris, positive exercise test and normal coronary angiogram) and eight healthy women (mean age 58 +/- 5 years) in a placebo-controlled, double-blind crossover trial. Two exercise tests were performed on separate days, separated by at least 1 week, after application of placebo or 100 micrograms/24 h estradiol-17-beta. In the control group there was no difference between estradiol and placebo. Patients with syndrome X, on the other hand, showed an increased time to angina (323 +/- 99 versus 233 +/- 67 s, P = 0.0044), time to 1 mm ST depression (257 +/- 142 versus 187 +/- 122 s, P = 0.039), total exercise time (363 +/- 104 versus 323 +/- 85 s, P = 0.038), and working capacity (93 +/- 17 versus 89 +/- 15 W, P = 0.0086) during active treatment. In conclusion, estradiol-17-beta has a beneficial effect on myocardial ischemia in postmenopausal women with syndrome X and may be a useful therapeutic agent in this disease.

Reduction in thrombotic events with heparin-coated Palmaz-Schatz stents in normal porcine coronary arteries.

BACKGROUND: The use of stents improves the result after balloon coronary angioplasty. Thrombogenicity of stents is, however, a concern. In the present study, we compared stents with an antithrombotic coating with regular stents. METHODS AND RESULTS: Regular stents were placed in coronary arteries of pigs receiving no aspirin (group 1; n = 8) or aspirin over 4 weeks (group 2, n = 10) or 12 weeks (group 3, n = 9). Stents coated with heparin (antithrombin III uptake, 5 pmol/stent) were placed in 7 pigs that did not receive aspirin (group 4). The other animals received aspirin and coated stents with a heparin activity of 12 pmol antithrombin III/stent (group 5, n = 10) or 20 pmol/stent (group 6, n = 10; group 7, n = 10). Quantitative arteriography was performed at implantation and after 4 (groups 1, 2, and 4 through 6) or 12 weeks (groups 3 and 7). In an additional 5 animals, five regular and five coated stents (20 pmol/stent) were placed and explanted after 5 days for examination of the early responses to the implants. Thrombotic occlusion of the regular stent occurred in 9 of 27 in groups 1 through 3. However, in 0 of 30 of the animals receiving high-activity heparin-coated stents (groups 5 through 7), thrombotic stent occlusion was observed (P < .001). Histological analysis at 4 weeks showed that the neointima in group 6 was thicker compared with its control group 2 (259 +/- 104 and 117 +/- 36 microns, P < .01), but at 12 weeks the thickness was similar (152 +/- 61 and 198 +/- 49 microns, respectively). Comparison at 5 days suggested delayed endothelialization of the coating. CONCLUSIONS: High-activity heparin coating of stents eliminates subacute thrombosis in porcine coronary arteries.

Melanoma cell destruction in the microvasculature of perfused hearts is reduced by pretreatment with vitamin E.

Different mechanisms have been proposed to explain the rapid elimination of circulating malignant cells: interactions with circulating leukocytes, mechanical trauma induced by deformation, shear forces and tissue pressure variations. Based on earlier observations in an isolated heart perfusion model the present study was performed to test whether or not microvascular damage of malignant cells depends on their anti-oxidant status. Murine melanoma B16F10 cells, pretreated with 100 microM alpha-tocopherol (or solvent) for 48 h, were used. The cells were perfused into the coronary vasculature of isolated hearts from C57/BL6 mice. Passing cells were collected and their viability determined by Trypan Blue exclusion. The hearts were processed for electron microscopy and the frequency of ultrastructurally intact and damaged B16 cells trapped in capillaries was recorded. In filter perfusion experiments the effect of vitamin E pretreatment on the resistance of the melanoma cells to mechanical deformation was determined. Morphometrically, cell size and cell profile perimeter excess of the melanoma cells were computed. Vitamin E pretreatment increased perfused cell viability from 50% to 81%. Ultrastructurally 30% of the intracapillary vitamin E treated cells were damaged (plasmalemmal fragmentation or worse) as compared to 58% of control cells. These differences were statistically significant (P < 0.01) whereas no differences could be demonstrated in filterability, cell size, or cell surface excess. The data support the hypothesis that malignant cell destruction in the systemic microcirculation is at least partly dependent on an oxygen metabolite mediated process, the exact nature (e.g. superoxide, hydrogen peroxide, nitric oxide) of which remains to be determined.

Partitioning of proteins in dextran/hydrophobically modified dextran aqueous two-phase systems.

Partitioning of proteins was studied in aqueous two-phase systems composed of the polymers dextran and hydrophobically modified dextran. The modified dextrans were benzoyl dextran with a degree of substitution of 0.17 and valeryl dextran with a degree of substitution of 0.20. Phase diagrams for the systems of dextran/benzoyl dextran and dextran/valeryl dextran were determined at room temperature. The proteins studied were beta-galactosidase, bovine serum albumin, beta-lactoglobulin, lysozyme, myoglobin and cytochrome C. The partition coefficients of a series of salts were determined in dextran/benzoyl dextran two-phase systems. The addition of salts had strong effect on the partitioning of proteins. This effect was related to protein net charge and the position of the ions in the Hofmeister series. Cross partitioning of bovine serum albumin was studied in a dextran/benzoyl dextran aqueous two-phase system.

The structure and function of the chloroplast photosynthetic membrane - a model for the domain organization.

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14-18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.

Evidence for two different Rieske iron-sulfur proteins in the cytochrome bf complex of spinach chloroplast.

Spinach thylakoids, grana vesicles, stroma lamellae vesicles and also isolated cytochrome bf complex, were analysed by two-dimensional polyacrylamide gel electrophoresis employing isoelectric focusing in the first dimension and sodium dodecyl-sulfate polyacrylamide gel electrophoresis in the second dimension. About 100 thylakoid membrane proteins were resolved. In all cases the Rieske FeS protein separated into two polypeptide spots having the isoelectric points of 5.1 and 5.4, respectively. The Rieske FeS protein was identified by immunoblot analysis and by microsequences of the first 23 N-terminal amino acids. The intensity of the Coomassie brilliant blue stain of the two spots was stronger for the Rieske FeS protein of the grana vesicles than for that of the stroma lamellae vesicles.

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing.

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the α and ß subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75-5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.

Characterization of a non-detergent PS II-cytochrome b/f preparation (BS).

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (-196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249-259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8-2.0, have a high oxygen evolving capacity (295 µmol O2 per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6-7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.

Preparation of benzoyl dextran and its use in aqueous two-phase systems.

The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.

The domain organization of the plant thylakoid membrane.

A model of the photosynthetic membrane from higher plants is presented. The different photosystems, PSI alpha, PSI beta, PSII alpha and PSII beta, are located in separate domains. The photosystems with the largest antenna systems, the alpha systems, are in the grana and the other in the stroma lamellae. In each grana disc PSI alpha is located in a flat annulus surrounding a circular PSII alpha domain. In this the PSII alpha units with the largest antennae are found in the center. The model is consistent with results from recent membrane fractionation experiments.

Separation processes in biotechnology. Aqueous two-phase separations.

Aqueous two-phase systems are useful for separation of a wide range of water-compatible substances (from peptides to cells). The selectivity of the separation normally increases with the size of the partitioned molecules or particles. The partition and separation capacity can be influenced in a number of ways, including electric charge, hydrophobicity, or specific ligand binding. Because of the simpleness in operation and high capacity, aqueous two-phase systems are well suited for large-scale purification of biomaterials such as enzymes and other specific proteins.

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems.

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.

Preparation of highly enriched photosystem II membrane vesicles by a non-detergent method.

An improved, non-detergent, method for preparative isolation of PS II membrane vesicles from spinach chloroplasts is presented. Thylakoids (chlorophyll (Chl) a/b ratio 2.8, Chl/P700 435) were fractionated by Yeda press treatment and aqueous two-phase partition to yield inside-out vesicles (1) (chl a/b 2.2, chl/P700 700). These vesicles were subjected a sonication - phase partitioning procedure; steps of sonication of inside-out vesicles, while still present in a dextran-polyethylene glycol two-phase system were alternated by phase partition. These steps selectively removed P700-containing membrane fragments from the inside-out vesicles and yielded a membrane fraction with improved PS II purity (Chl a/b ratio 1.9, Chl/P700 1500) and retained oxygen evolving capacity (295 µmol O2 mg Chl(-1) h(-1)).

Affinity separation of proteins in aqueous three-phase systems.

Aqueous polymer three-phase systems composed of dextran-Ficoll-polyethylene glycol-water have been used for affinity partition of proteins. The upper, middle, and lower phases are rich in polyethylene glycol, Ficoll, and dextran, respectively. Affinity partition was performed using the reactive dyes Cibacron Blue F36-A and Remazol Yellow GCL which are known as specific ligands for albumin and prealbumin from human serum. When the ligands were bound alternatively to polyethylene glycol, Ficoll, or dextran the target proteins were directed toward the upper, middle, or lower phase, respectively. In the presence of two ligands immobilized to two different polymers the distribution of two proteins could be steered to different phases at the same time. Serum albumin and prealbumin could be separated by using Cibacron Blue-Ficoll and Remazol Yellow-dextran or Cibacron Blue-polyethylene glycol and Remazol Yellow-dextran as polymer ligands.

Counter-current distribution of sonicated inside-out thylakoid vesicles.

Inside-out thylakoid vesicles were isolated from spinach chloroplasts, and fragmented by sonication. Different fragments were separated by counter-current distribution and analyzed for chlorophyll and P700. The inside-out vesicles had a chlorophyll a/b ratio of 2.2-2.4 (original chloroplasts 2.8-3.0). After further fragmentation of the inside-out vesicles by sonication and separation by countercurrent distribution three populations of vesicles were obtained having chlorophyll a/b ratios of 1.7, 1.9 and 2.5 respectively. The P-700 was depleted in fractions with lower chlorophyll a/b ratio and was nearly absent in the fraction having a chlorophyll a/b ratio of 1.7 (chlorophyll/P700 greater than 4500 mol/mol). That PSII membrane vesicles, with such a low chlorophyll a/b ratio and lacking PSI, can be prepared by a non-detergent method provides strong support for the notion that PSI and PSII are segregated along the thylakoid membrane. A plot of P700 per chlorophyll against chlorophyll b/(a + b) fits a straight line connecting the pure PSI membrane (chlorophyll a/b = 6; P700/chlorophyll = 5.6 mmol/mol) with the pure PSII membrane (chlorophyll a/b = 1.7; P700 = 0). These two membranes can be considered as separate phases of a two-dimensional phase system. Models for the thylakoid membrane are discussed.