Tumor angiogenesis induced by granulocyte chemotactic protein-2 as a countercurrent principle.

Chemokine production by tumors is a well-known phenomenon, but its role in tumor biology remains debatable. Although intratumoral injection of granulocyte chemotactic protein-2 (GCP-2) had no effect on tumor parameters, needle-free stable expression of the chemokine resulted in enhanced tumor growth. It is shown here that tumors that express a potent form of GCP-2 induce a strong influx and activation of tumor-associated neutrophils. The production of GCP-2 leads to intratumoral expression of gelatinase B and advantage for tumor growth by increased angiogenesis. These results are in line with the countercurrent principle of chemokine action and support the notion that paraneoplastic expression of ELR-positive CXC chemokines has to be blocked rather than stimulated in cancer therapy.

Three-factor response surface optimization of the production of dextran dextrinase by Gluconobacter oxydans.

Response surface methodology (RSM) and a five-level three-factor central composite rotatable design (CCRD) were used to evaluate the effect of glycerol and peptone concentration and initial pH on dextran dextrinase (DDase) production by Gluconobacter oxydans. Optimal fermentation conditions were 20.59 g/l of glycerol, 6.67 g/l of mycological peptone and an initial pH of 6.14. The predicted DDase yield of the optimised fermentation was 0.207 U/ml, whereas an actual experimental yield of 0.208 +/- 0.025 U/ml was obtained.

Characterization of the efficiency of a cell separation process by the extent of elimination of a contaminating cell type.

A stepwise approach to the selection of an appropriate technique for a cell separation problem is presented in which the preparative purification of cells is linked to their analytical separation. We have introduced the extent of elimination of a contaminating cell type from the cell type which one chooses to purify, as a separation parameter that characterizes the efficiency of a separation process independently of the relative cell composition of the starting material. In order to compare different separation techniques, a preparative fraction boundary needs to be chosen between the cell types. We defined this boundary in terms of the physical property on which the separation is based such that yield and purity of the isolated cell suspension are optimized simultaneously. With this analytical approach, it was found that a similar elutriation technique separated human and equine mononuclear cells equally well and that the separability of human monocytes and lymphocytes improved when the cells were separated by increasing the limiting sedimentation coefficient value of the elutriation chamber in small increments.

Interaction between the respiratory burst activity of neutrophil leukocytes and experimentally induced Escherichia coli mastitis in cows.

The respiratory burst activity of neutrophil leukocytes from bovine peripheral blood was studied before and during an experimentally induced Escherichia coli mastitis. The competence of neutrophils to generate reactive oxygen species following stimulation with opsonized particles prior to infection was negatively correlated with severity of subsequently induced E. coli mastitis. In the presence of the soluble activator, phorbol myristate acetate, no such correlation was obtained. However, combination of blood neutrophil numbers with phorbol myristate acetate induced respiratory burst competence, called reactive oxygen species-generating capacity, displayed a negative correlation with the intensity of a subsequent inflammation of the bovine mammary gland. At the onset of mastitis, a concomitant reduction in blood neutrophil numbers, a strong shift in cell types, and a substantial decrease in production of reactive oxygen species occurred. Reestablishment and even enhancement of the respiratory burst activity coincided with the reappearance of mature neutrophils. Possible stimulatory effects on neutrophil superoxide generation are discussed. Data suggest that generation of reactive oxygen species by mature neutrophils may be of primary importance for microbial killing during the onset and recovery from mastitis.

Activation of a NADPH oxidase from horse polymorphonuclear leukocytes in a cell-free system.

A postnuclear cell fraction from resting horse polymorphonuclear (PMN) leukocytes incubated with fatty acid-salt ions such as oleate or linoleate generated a NADPH-dependent oxygen consumption and superoxide production. Oxidative activity was negligible or absent in the postnuclear fraction from mononuclear leukocytes, p-chloromercuribenzene sulfonic acid-treated granulocytes, and granulocytes from a patient with chronic granulomatous disease. Although consistently associated with the membrane fraction from resting PMN leukocytes, the superoxide-generating activity was shown to be dependent on a thus far unknown cytosolic constituent. The apparent Km's for NADPH and NADH (66 and 1,600 microM, respectively), the pH optimum for the reaction (7.0), the cyanide insensitivity, and transient nature of the reaction together with the stoichiometric relationship between oxygen uptake and NADPH oxidation led to the conclusion that in the presence of cytosol a cell-free latent respiratory burst oxidase can be converted into an active enzyme by interaction with oleate micelles.

Subcellular distribution of particle-associated enzymes in horse neutrophil leukocytes.

The subcellular components of purified neutrophil leukocytes from horse blood were fractionated by isopyknic equilibration in sucrose and metrizamide gradients. Five classes of particles have been identified: dense azurophil granules containing the bulk of the lysosomal acid hydrolase and peroxidase activity (A); less dense particles, containing all the lysozyme activity, but not resolved from a second population of azurophils B, and particles of low density, biochemically characterized as a plasma membrane fraction (C). Isopyknic equilibration in sucrose disclosed a minor membrane fraction (D) of unidentified origin, whereas mitochondria were best resolved on metrizamide gradients.

Acid phosphatase heterogeneity in horse neutrophil and eosinophil leukocytes.

Two distinct groups of acid phosphatase containing granules were characterized in neutrophils, each group displaying different multiple forms of the enzyme. The heavy granule acid phosphatase showed a lysosomal location. A second lighter group of particles contained a thermolabile, thiol-dependent acid p-nitrophenyl and alpha-naphtylphosphatase, an enzyme clearly different from lysosomal acid phosphatase. Acid phosphatase activity from eosinophil leukocytes appeared to be totally associated with the typical eosinophil granules. On mechanical disruption of these particles, an acid phosphatase was released which differed in substrate and inhibitor specificity, in electrophoretic pattern, and in thermosensitivity, from the remaining matrix-bound enzyme.