Josephine Contrino

In situ detection of tissue factor in vascular endothelial cells: correlation with the malignant phenotype of human breast disease.

Expression of tissue factor (TF) in the endothelium has been observed only rarely in human disease and has been thought to be elaborated on the surface of vascular endothelial cells (VECs) in vitro as an artifact of tissue culture. Using monoclonal antibodies and a novel probe for functional TF, we have localized TF to the VECs (and tumor cells) within the tumors of seven patients with invasive breast cancer but not in the VECs (or tumor cells) of benign tumors from ten patients with fibrocystic disease of the breast. The potent procoagulant TF was shown to be a marker of the initiation of angiogenesis in human breast cancer. Further evidence that the TF was the demonstration of a similar distribution of cross–linked fibrin only in the VECs of the malignant tumors. We interpret these data as further support for the concept that tumor cells can activate nearby VECs and regulate blood vessel growth in vivo. Large clinicalpathologic studies will be necessary to determine whether TF is a useful marker for the “switch to the angiogenic phenotype” in human breast disease and/or correlates with the thromboembolic complications of breast cancer.

Tissue factor expression in human leukemic cells

Patients with acute leukemia are at increased risk for thrombotic and hemorrhagic complications, particularly those patients with acute promyelocytic leukemia (APL) undergoing induction chemotherapy. These serious complications have been attributed by some authors to the release of tissue factor (TF) procoagulant activity (PCA), particularly during cytotoxic chemotherapy. In previous studies of normal peripheral blood cells, only cells of the monocyte lineage have been found to express TF PCA. Therefore, several questions remain regarding the origin and characterization of the PCA in malignant leukemic cells, particularly those thought to be derived from granulocyte progenitor cells. We utilized a full-length cDNA probe, several monoclonal antibodies (MAbs) and a sensitive one-stage PCA assay to study the expression of TF in the human cell line, HL-60, in human peripheral blood monocytes/macrophages (Mo/Mø) and in highly purified populations of human polymorphonuclear leukocytes (PMN). In the HL-60 cells we detected low but significant levels of TF mRNA and TF antigen (TF:Ag). In unstimulated cells, coordinate increased levels of TF mRNA, TF:Ag and TF PCA expression were noted following phorbol-ester-induced macrophage differentiation of the cells, but a decreased level of TF mRNA with no change in the basal level of TF:Ag expression occurred following retinoic acid-induced granulocyte differentiation of this cell line. Long-term cultures of stimulated mature Mo/Mø demonstrated initial coordinate expression of TF mRNA, TF:Ag and TF PCA, but TF:Ag expression persisted even after 7 days (when TF PCA was undetectable). No TF PCA, TF:Ag or TF mRNA was demonstrated in highly purified populations of human PMN, regardless of culture conditions. Discordant expression of TF mRNA, TF:Ag and TF PCA in HL-60 cells suggests the possibility of novel, post-synthetic mechanisms for the regulation of TF PCA expression, which might be dependent on the phenotypic differentiation level of the cell. Such mechanisms (yet to be defined) might account for the ability of some leukemic cells, which frequently express characteristics of more than one cell line (e.g. monocytes and granulocytes), to express a TF gene product capable of activating blood coagulation.

Murine model of otitis media with effusion: Immunohistochemical demonstration of il-1α antigen expression

Recent studies have suggested that cytokines likely play a central role in the formation and maintenance of otitis media with effusion (OME). Currently, there is no immunologically defined animal model for the study of cytokines as they contribute to the formation of OME. In the present study, a murine model of OME, using eustachian tube blockage via an external surgical approach, was developed. The murine model temporal bone histology appears to mimic the histology found in chronic otitis media with effusion in humans. Additionally, using this murine model, interleukin‐1α (IL‐1α) expression was detected in the middle ear using standard immunohistochemical techniques. IL‐1α seemed localized to the epithelial lining of the middle ear as well as 5% to 10% of inflammatory cells. This model should provide the necessary tool to further study the immunologic aspects of OME.

Elevated interleukin-1 expression in human neonatal neutrophils

ABSTRACT: Newborn infants have an increased morbidity and mortality from infection caused in part by diminished polymorphonuclear leukocyte (PMN) function and impaired recruitment of PMN to sites of inflammation. Recent studies in our laboratory and others have demonstrated the in vitro expression of several cytokines, including IL-1-β, in adult human peripheral blood PMN. Because newborn infants have an impaired inflammatory response, we sought to compare the synthetic capability and regulation of cytokine expression in neonatal and adult PMN. In our present studies, we found that tumor necrosis factor-α and lipopolysaccharide could induce IL-1-β expression in both neonatal and adult PMN and that neonatal PMN produced significantly more IL-1-β when compared with adult PMN. The PMN chemotactic peptide fMet-Leu-Phe did not induce IL-I-β expression in either adult or neonatal PMN. Elevated cytokine expression by neonatal PMN may play an important role in the regulation of the immune and inflammatory systems at sites of injury and infection in neonates.

The in vitro effects of mercury on peritoneal leukocytes (PMN and macrophages) from inbred brown Norway and Lewis rats

The present paper demonstrates that HgCl2 can affect rat peritoneal polymorphonuclear leukocyte (PMN) and macrophage (M phi) functions in vitro. In addition, we have noticed that these effects of mercury vary according to the rat strain: for example, HgCl2 stimulates H2O2 release from Lewis (LEW) but not Brown Norway (BN) PMN. Similarly, LEW M phi produce high levels of H2O2 when exposed to HgCl2 in vitro, whereas BN M phi do not. Finally, mercury inhibits erythrophagocytosis of both LEW and BN resident peritoneal M phi. Preliminary experiments using M phi from other rat strains have also shown that MAXX M phi are stimulated by HgCl2 to release H2O2 in vitro, whereas Yoshida M phi are inhibited. Differences in lymphocyte responses (e.g. delayed-type hypersensitivity reactions and mitogen stimulation) between rats of various strains are well known. To these examples one may now add variations in PMN and M phi responses to mercury and possibly other metals. Our results suggest that caution should be exercised in interpreting the outcome of immunotoxicity studies in experimental animals. In particular, outbred rats may not provide appropriate models, that might be better obtained by comparative investigations of rats from various inbred strains.

Modulation of Neonatal Neutrophil Function by Pentoxifylline

ABSTRACT: Immunomodulating agents are being investigated for treatment of infection in newborn infants where morbidity and mortality remain high despite the continued development of new antibiotics. We studied the effect of the methylxanthine pentoxifylline on polymorphonuclear leukocyte (PMN) chemotaxis, F-actin content, and phagocytic activity as measured by nitroblue tetrazolium reduction and H2O2 production in neonates and adults to determine whether pentoxifylline might be useful in augmenting PMN function. The drug was found to have a dose-dependent effect on both neonatal and adult PMN function with enhancement at lower concentrations and suppression at higher concentrations. PMN chemotaxis increased 42% (p < 0.01) in neonates and 16% (p < 0.05) in adults at 100 μg/mL of pentoxifylline and it decreased 4 and 25%, respectively, at 4000 μg/mL. PMN nitroblue tetrazolium reduction increased by 34% in neonates and 23% (p < 0.05) in adults at 100 μg/mL of pentoxifylline and decreased by 52 (p < 0.01) and 74% (p < 0.01), respectively, at 2000 μg/mL. Similar dose-dependent responses were noted with F-actin content and H2O2 production. These and other observations support the hypothesis that pentoxifylline has a broad range of effects on PMN but that a primary effect is alteration of PMN deformability. Pentoxifylline has potential clinical use as an immunomodulator in augmenting impaired PMN function in neonates and other immunocompromised hosts or in suppressing excessive PMN activity in certain disease processes.

Silica Induced Suppression of the Production of Third and Fifth Components of the Complement System by Human Lung Cells In Vitro

Although investigations to date have demonstrated the ability of the monocyte/macrophage to synthesize complement components, only a limited number of studies on complement synthesis by nonhepatic tissue cells have been reported. To begin to fill this gap in our knowledge we have recently evaluated the ability of lung tissue cells to synthesize and secrete various complement components in vitro. Using 35S-methionine incorporation and immunoprecipitation techniques we have previously demonstrated the ability human lung type II pneumocytes (A549) and human lung fibroblasts (WI-38), to synthesize and secrete a variety of both early and terminal complement components, as well as several regulatory proteins including Clr, Cls, C4, C3, C5, C6, C7, C8, C9, Factor B, Factor H, Factor I and Cls inactivator. Our present studies demonstrate the capability of silica to regulate complement component production by A549 cells, but not complement component production by WI-38 cells. Specifically, using sensitive ELISAs we demonstrated that a non-toxic dose of silica had the capability to suppress the production of both C3 and C5 by A549 pneumocytes by 40-50 percent, but had no effect on C3 or C5 synthesis by WI-38 fibroblasts. Additionally, using 35S-methionine incorporation and TCA precipitation techniques, we demonstrated that suppression of C3 and C5 production by silica treated A549 pneumocytes was not a result of suppression of total protein synthesis. These studies demonstrate that silica, which has been implicated in pulmonary diseases, has the capability to regulate local complement production by lung tissue cells in vitro. In vivo, this suppression of complement production by the type II pneumocytes could alter the local tissue reservoir of complement components during infection and pulmonary injury, thus resulting in depressed pulmonary host defense.