Shastri Sookhai

Taurolidine Inhibits Tumor Cell Growth In Vitro and In Vivo

AbstractBackground: Taurolidine, a derivative of the amino acid taurine, exhibits antiendotoxin, antibacterial, and antiadherence activity. We hypothesized that Taurolidine may inhibit tumor cell growth, both in an in vitro and in vivo setting. Our aim was to examine the effect of Taurolidine on the growth of a rat metastatic colorectal tumor cell line (DHD/K12/TRb) in vitro and in vivo. Methods: In the in vitro experiments, DHD/K12/TRb cells were incubated with 5, 10, 15, 25 μg/ml of Taurolidine. Cells incubated in culture medium alone were used as controls. Cell proliferation, cell viability, cell death, and cell apoptosis were measured using commercially available techniques. In the in vivo experiment, BD IX rats were randomized into two groups (n = 10/group). Group A (control) underwent laparotomy and instillation of DHD/K12/TRb tumor cells intraperitoneally followed by phosphate buffered saline (PBS). Group B received Taurolidine (100 mg/kg) instead of PBS. Animals were killed after 24 days and tumor burden assessed by counting the number of tumor nodules in the peritoneal cavity. Results: Incubation of the tumor cells with Taurolidine resulted in a 4-fold decrease in proliferation rates (25 ± 4% vs. 100 ± 28% for controls) and a 4-fold increase in cell necrosis as demonstrated by the increase in LDH release (403 ± 28% vs. 100 ± 26% for controls), at a Taurolidine concentration of 25 μg/ml. A dose-dependent decrease in cell viability was also observed. In the in vivo study, local Taurolidine administration resulted in significant decreases in tumor burden (3 ± 1 nodules in Group B animals vs. 649 ± 101 nodules in Group A animals). Conclusions: Taurolidine inhibits the growth of a rat metastatic colorectal tumor cell line in vitro and in vivo and thus may have potential in the prevention of peritoneal metastases.

A Novel Therapeutic Strategy for Attenuating Neutrophil-mediated Lung Injury In Vivo

ObjectiveTo evaluate the effect of inhalation of aerosolized opsonized dead Escherichia coli on inflammatory pulmonary neutrophil (PMN) apoptosis, lung injury, and survival in a PMN-mediated lung injury model in vivo. Summary Background DataNeutrophils that have transmigrated into an inflammatory focus display increased functional capacity and delayed apoptosis, resulting in an increased capacity to injure normal host tissue. The authors have previously shown that E. coli induces PMN apoptosis in vitro. MethodsLung injury mediated by PMNs was established by aortic occlusion and reperfusion. Adult male Sprague-Dawley rats were randomized into four groups: sham ischemia–reperfusion (I/R) treated with intratracheal inhalation of aerosolized normal saline, I/R treated with aerosolized normal saline intratracheally, I/R treated with aerosolized opsonized dead E. coli intratracheally, and I/R treated with aerosolized opsonized dead E. coli and the caspase inhibitor zVAD-FMK intratracheally 5 minutes before reperfusion. Both systemic and bronchoalveolar lavage PMNs were isolated and apoptosis was quantified at 0, 6, 12, 18, and 24 hours. Lung injury parameters including wet/dry lung weight ratio, histology, myeloperoxidase activity, and protein content were also assessed. In addition, a survival study was performed, both in a prophylactic and in a therapeutic setting. ResultsAdministration of aerosolized dead E. coli before the reperfusion injury induced pulmonary PMN apoptosis and reversed the delayed apoptosis evident in the I/R plus normal saline group. There was also a significant improvement in lung injury parameters as well as in survival, both prophylactically as well as therapeutically. ConclusionsDirectly modulating PMN cell death represents a novel mechanism for attenuating PMN-mediated lung injury and may ultimately benefit the outcome in patients with adult respiratory distress syndrome.

Dopamine attenuates the chemoattractant effect of interleukin-8: a novel role in the systemic inflammatory response syndrome.

Activated neutrophil (PMN) adherence to vascular endothelium comprises a key step for both transendothelial migration and initiation of potentially deleterious release of PMN products. The biogenic amine, dopamine (DA), has been used for several decades in patients to maintain hemodynamic stability. The effect of dopamine on PMN transendothelial migration and adhesion receptor expression and on the endothelial molecules, E-selectin and ICAM-1, was evaluated. PMN were isolated from healthy controls, stimulated with lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) and treated with dopamine. CD 11b and CD 18 PMN adhesion receptor expression were assessed flow cytometrically. In a separate experiment, the chemoattractant peptide, IL-8, was placed in the lower chamber of transwells, and PMN migration was assessed. Human umbilical vein endothelial cells (HUVEC) were stimulated with LPS/TNF-alpha and incubated with dopamine. ICAM-1 and E-selectin endothelial molecule expression were assessed flow cytometrically. There was a significant increase in transendothelial migration in stimulated PMN compared with normal PMN (40 vs. 14%, P < 0.001). In addition, PMN CD11b/CD18 was significantly upregulated in stimulated PMN compared with normal PMN (252.4/352.4 vs. 76.7/139.4, P < 0.001) as were endothelial E-selectin/ICAM-1 expression compared with normal EC (8.1/9 vs. 3.9/3.8, P < 0.05). After treatment with dopamine, PMN transmigration was significantly decreased compared with stimulated PMN (8% vs. 40%, P < 0.001). Furthermore, dopamine also attenuated PMN CD11b/CD18 and the endothelial molecules E-selectin and ICAM-1 compared with stimulated PMN/EC that were not treated dopamine (174/240 vs. 252/352, P < 0.05 and 4/4.4 vs. 8.1/9, P < 0.05. respectively). The chemoattractant effect of IL-8 was also attenuated. These results identify for the first time that dopamine attenuates the initial interaction between PMN and the endothelium, and consequently, modulates PMN exudation. Thus, biogenic amines, including dopamine, may function as anti-inflammatory cytokines.

Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro.

Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS). Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer. PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens. The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity. We found that BLP-activated human PMN as evidenced by increased CD11b/CD18 (Mac-1) expression. Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14). A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected. PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMNs or HUVEC independently with BLP. This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70. BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli. BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.

Attenuation of Pancreatitis-Induced Pulmonary Injury by Aerosolized Hypertonic Saline

BACKGROUND The immunomodulatory effects of hypertonic saline (HTS) provide potential strategies to attenuate inappropriate inflammatory reactions. This study tested the hypothesis that administration of intratracheal aerosolized HTS modulates the development of lung injury in pancreatitis. METHODS Pancreatitis was induced in 24 male Sprague-Dawley rats by intraperitoneal injection of 20% L-arginine (500 mg/100 g body weight). At 24 and 48 h, intratracheal aerosolized HTS (7.5% NaCl, 0.5 mL) was administered to 8 rats, while a further 8 received 0.5 mL of aerosolized normal saline (NS). At 72 hours, pulmonary neutrophil infiltration (myeloperoxidase activity) and endothelial permeability (bronchoalveolar lavage and wet:dry weight ratios) were assessed. In addition, histological assessment of representative lung tissue was performed by a blinded assessor. In a separate experiment, polymorphonucleocytes (PMN) were isolated from human donors, and exposed to increments of HTS. Neutrophil transmigration across an endothelial cell layer, VEGF release, and apoptosis at 1, 6, 12, 18, and 24 h were assessed. RESULTS Histopathological lung injury scores were significantly reduced in the HTS group (4.78 +/- 1.43 vs. 8.64 +/- 0.86); p < 0.001). Pulmonary neutrophil sequestration (1.40 +/- 0.2) and increased endothelial permeability (6.77 +/- 1.14) were evident in the animals resuscitated with normal saline when compared with HTS (0.70 +/- 0.1 and 3.57 +/- 1.32), respectively; p < 0.04). HTS significantly reduced PMN transmigration (by 97.1, p = 0.002, and induced PMN apoptosis (p < 0.03). HTS did not impact significantly upon neutrophil VEGF release (p > 0.05). CONCLUSIONS Intratracheal aerosolized HTS attenuates the neutrophil-mediated pulmonary insult subsequent to pancreatitis. This may represent a novel therapeutic strategy.