Taurine modulates neutrophil function but potentiates uropathogenic E. coli infection in the murine bladder.

Eradication of a urinary tract infection (UTI) appears to be related to a number of innate host defence mechanisms and their interactions with invading bacteria. Recurrent UTIs (rUTIs) pose a difficult problem in that these bacteria use both host and bacterial factors to evade elimination. Neutrophil bactericidal function is depressed, both systemically and in urine, in patients with a history of recurrent UTI. Taurine is a semi-essential amino acid and is successful in preserving neutrophil bactericidal function in urine. Taurine may preserve neutrophil function at the urothelium and thus aid UTI resolution. Adult female (6 weeks old) C57Bl/6 mice were randomised into three groups: a saline gavage only control group, a saline gavage + E. coli group, and a taurine gavage + E. coli group [21 g/70 kg taurine in 0.9% normal saline (N/S) for 5 days]. Whilst taurine gavage pre-treatment resulted in increased serum neutrophils respiratory burst activity, at the urothelial-endothelial interface it caused higher colony forming units in the urine and a higher incidence of E. coli invasion in the bladder wall with no evidence of increased bladder wall neutrophils infiltration on MPO assay of histological assessment. Histologically there was also evidence of reduced bladder inflammation and urothelial cell apoptosis. In conclusion, taurine effectively increases neutrophils activity but given its anti-inflammatory properties, at the expense of decreased urothelial-endothelial activation thus preventing clearance of active E. coli infection in the bladder. Despite the negative results, this study demonstrates the importance of modulating interactions at the urothelial interface.

Therapeutic vaccination with dendritic cells pulsed with tumor-derived Hsp70 and a COX-2 inhibitor induces protective immunity against B16 melanoma.

Prophylactic immunization of mice with autologous tumor-derived heat shock proteins (Hsp) generates effective anti-tumor immunity. However, this approach is ineffective when used therapeutically, partly due to the immunosuppressive effects of the growing tumor. Here we sought to overcome this problem by therapeutic vaccination with dendritic cells (DC) pulsed with Hsp70 and a COX-2 inhibitor. We found that Hsp70 induces IL-6 and IL-10 production and suppressed expression of CD40 on DC. Incubation of DC with tumor-conditioned medium attenuated Hsp70-induced expression of CD80 and induced expression of COX-2. Inhibition of COX-2 partially reversed the stimulatory effect of Hsp70 on DC IL-6 and IL-10 production and enhanced expression of CD80 and MHC classes I and II. Therapeutic administration of DC pulsed in vitro with Hsp70 in the presence of a COX-2 inhibitor significantly reduced progression of B16 tumors in mice and significantly enhanced survival. This was associated with a reduction in the frequency of IL-10-producing CD4(+) T cells and enhancement of IFN-gamma-producing CD8(+) T cells. Our findings provide a novel immunotherapeutic approach against cancer based on attenuation of COX-2-mediated immunosuppression using in vitro modulated DC.

Attenuating regulatory T cell induction by TLR agonists through inhibition of p38 MAPK signaling in dendritic cells enhances their efficacy as vaccine adjuvants and cancer immunotherapeutics.

TLR ligands are potent adjuvants and promote Th1 responses to coadministered Ags by inducing innate IL-12 production. We found that TLR ligands also promote the induction of IL-10-secreting regulatory T (Treg) cells through p38 MAPK-induced IL-10 production by dendritic cells (DC). Inhibition of p38 suppressed TLR-induced IL-10 and PGE(2) and enhanced IL-12 production in DC. Incubation of Ag-pulsed CpG-stimulated DC with a p38 inhibitor suppressed their ability to generate Treg cells, while enhancing induction of Th1 cells. In addition, inhibition of p38 enhanced the antitumor therapeutic efficacy of DC pulsed with Ag and CpG and this was associated with an enhanced frequency of IFN-gamma-secreting T cells and a reduction of Foxp3(+) Treg cells infiltrating the tumors. Furthermore, addition of a p38 inhibitor to a pertussis vaccine formulated with CpG enhanced its protective efficacy in a murine respiratory challenge model. These data demonstrate that the adjuvant activity of TLR agonists is compromised by coinduction of Treg cells, but this can be overcome by inhibiting p38 signaling in DC. Our findings suggest that p38 is an important therapeutic target and provides a mechanism to enhance the efficacy of TLR agonists as vaccine adjuvants and cancer immunotherapeutics.

Fracture hematoma is a potent proinflammatory mediator of neutrophil function.

BACKGROUND: Patients with multiple skeletal injuries are susceptible to acute respiratory distress syndrome and multiple organ failure, which result from hyperactivation of the immune system. This study was designed to evaluate in vitro the proinflammatory properties of fracture hematoma (FH). METHODS: FH was isolated from patients undergoing emergent open reduction and internal fixation for isolated closed fractures. Neutrophils (PMNs), isolated from healthy volunteers, were exposed to the FH supernatant and activation was examined (CD11b and CD18 adhesion receptor expression and respiratory burst). PMN phagocytosis, apoptosis, and transmigration across an endothelial barrier were also assessed. RESULTS: FH increased PMN respiratory burst (control, 100; FH-treated, 186) and phagocytosis (control, 100; FH-treated, 172) but had no effect on adhesion receptor expression. Transendothelial migration of PMNs was unaffected, although FH was toxic to endothelial cells. In contrast, apoptosis of FH-treated PMNs was delayed (control, 46; FH-treated, 8). CONCLUSION: These effects, although beneficial at the site of injury in the context of antibactericidal function, may cause PMN-mediated tissue injury systemically.

Taurine protects against PMN dysfunction and death in urine.

The polymorphonuclear neutrophil (PMN) is the primary pro-inflammatory cell in the host response to bacterial infection and, as the first line of defence, is the principal cell responsible for the recognition, phagocytosis and killing of bacteria. PMN function is known to be defective in the urine. High osmolarity is physiologic in the urine and this hypertonic environment has been shown to compromise neutrophil function. In this study, PMN function was found to be suppressed in urine. This correlated with significant cell death, both by apoptosis and necrosis. The amino acid taurine down regulated PMN cell death and preserved function in the urine, suggesting taurine as a therapeutic option for urinary tract infection.

N-acetylcysteine attenuates lung injury in a rodent model of fracture.

BACKGROUND: Neutrophil-mediated lung injury is a cause of significant morbidity and mortality in patients with multiple injuries. We have shown previously that fracture hematoma can activate neutrophils and is thus a putative mediator of the systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF) in those patients with severe skeletal trauma. Our aim was to establish a rodent model of fracture which caused lung injury and subsequently to administer a drug following fracture to attenuate the lung injury. The drug we chose was N-acetylcysteine, a potent antioxidant. ANIMALS AND METHODS: Adult Sprague-Dawley rats were assigned to 4 groups: (1) general anesthetic only, (2) general anesthetic with bilateral femur fractures and nailing, (3) general anesthetic and N-acetylcysteine, (4) general anesthetic with bilateral femur fractures and nailing and N-acetylcysteine after the injury (n = 6 in each group). The dose of N-acetylcysteine was 0.5 mg/kg which was given intraperitoneally after injury to the treated groups. The rats were killed 24 hours after injury and some parameters of lung injury were evaluated--i.e., bronchoalveolar lavage (BAL), lung tissue myeloperoxidase levels (MPO) and wet/dry ratios of lung tissue. The results were analyzed, using one-way analysis of variance. RESULTS: Bilateral femur fracture produced a significant lung injury, measured by increases in MPO (25-43 microg/g tissue) and BAL protein (460-605 microg/mL). This effect was attenuated by treatment with N-acetylcysteine (MPO 43-9 microg/mL, BAL protein 605-198 microg/mL). INTERPRETATION: N-acetyl cysteine, if given after skeletal trauma, is of potential therapeutic benefit, in preventing SIRS, ARDS and MOF.

The 90/90 pillow reduces blood loss after knee arthroplasty: a prospective randomized case control study.

Our goal was to evaluate the 90/90 pillow as an effective means of preventing blood loss after knee arthroplasty. We performed a prospective randomized case-control study on 40 patients undergoing knee arthroplasty. All patients were treated in a standard fashion, except for the use of the 90/90 pillow in one group. The 90/90 pillow flexes the knee to 90 degrees and is in situ for 24 hours after surgery. We measured blood loss at time points from 1 to 48 hours. Range of motion was assessed, as were analgesic requirements. Statistical analysis was performed on our results. We found a statistically significant reduction in blood loss without compromising range of motion. We advocate the 90/90 pillow after all knee arthroplasties.

Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies.

Tissue hypoxia occurs where there is an imbalance between oxygen supply and consumption. Hypoxia occurs in solid tumours as a result of an inadequate supply of oxygen, due to exponential cellular proliferation and an inefficient vascular supply. It is an adverse prognostic indicator in cancer as it is associated with tumour progression and resistance to therapy. The expression of several genes controlling tumour cell survival are regulated by hypoxia, e.g., growth factors governing the formation of new blood vessels, and hypoxia-responsive transcription factors modulating the expression of genes, which promote tumour cell survival. This review outlines some of the pathways by which tumour hypoxia leads to chemotherapeutic resistance, directly due to lack of oxygen availability, and indirectly due to alterations in the proteome/genome, angiogenesis and pH changes. Some innovative therapies are also detailed which may potentially minimise or eliminate these problems associated with targeting solid tumours.

Taurine attenuates calcium-dependent, Fas-mediated neutrophil apoptosis.

The pathway involved in Fas-mediated neutrophil apoptosis remains to be fully elucidated. We examined whether this pathway involved either oxygen-dependent or calcium-dependent mechanisms. We also investigated whether taurine, a powerful antioxidant and regulator of intracellular calcium fluxes, could inhibit Fas-mediated neutrophil apoptosis. Neutrophils were stimulated with Fas monoclonal antibody in the presence or absence of taurine. Fas receptor ligation resulted in significant neutrophil apoptosis at 18 h. Engagement of the Fas receptor rapidly resulted in a significant decrease in intracellular calcium. Apoptosis was inhibited and intracellular calcium levels were maintained in the presence of calcium ionophore A23187 or taurine. Fas ligation did not result in an increase in intracellular reactive oxygen species. We have demonstrated that Fas-mediated neutrophil apoptosis occurs after a decrease in intracellular calcium and is reactive oxygen intermediate independent. Furthermore, the amino acid taurine attenuates this pathway of neutrophil apoptosis by calcium regulation. This newly identified role of taurine in the inhibition of Fas-mediated neutrophil apoptosis may have significant implications for future manipulation of host pro-inflammatory cell function.

Infiltrating immune cells, but not tumour cells, express FasL in non-small cell lung cancer: No association with prognosis identified in 3-year follow-up.

Non-small cell lung cancer (NSCLC) remains a difficult disease to treat and independent prognostic markers other than tumour stage and histology have not emerged. The immune cell content of solid tumours has been associated with tumour regression and at times, tumour progression. The involvement of immune cells in prognosis of NSCLC is poorly described. Poor immune responses within solid tumours have been linked with tumour production of immunosuppressive cytokines. Tumour expression of FasL is thought to disarm responses through the transduction of a death signal in Fas-expressing T cells. The existence of the 'tumour counterattack' in vivo has been questioned. We undertook to measure T cell and macrophage infiltration of the tumour bed in NSCLC and report the association between immune cell content and prognosis in a limited, 3-year analysis of survival (n = 113). In addition we investigated FasL expression (n = 45). T cells and macrophages were found to frequently infiltrate lung tumours, albeit in small numbers. Generally there were more T cells infiltrating than macrophages. T cell and macrophage numbers were not associated with prognosis. Lung tumours were found not to express FasL, although occasional immune cells surrounding tumour cells were strongly positive. FasL expression was not associated with prognosis in this series. Thus, immune cells infiltrating NSCLC are not capable of suppressing tumour growth, nor are they associated with tumour progression. We report that lung tumours do not express the FasL, and that although some immune cells are FasL positive, this is not a reflection of general immune cell activation.

Activation-induced cell death: the controversial role of Fas and Fas ligand in immune privilege and tumour counterattack.

Activation-induced cell death (AICD) is the process by which cells undergo apoptosis in a controlled manner through the interaction of a death factor and its receptor. Programmed cell death can be induced by a number of physiological and pathological factors including Fas (CD95)-Fas ligand (FasL/CD95L) interaction, tumour necrosis factor (TNF), ceramide, and reactive oxygen species (ROS). Fas is a 48-kDa type I transmembrane protein that belongs to the TNF/nerve growth factor receptor superfamily. FasL is a 40-kDa type II transmembrane protein that belongs to the TNF superfamily. The interaction of Fas with FasL results in a series of signal transductions which initiate apoptosis. The induction of apoptosis in this manner is termed AICD. Activation-induced cell death and Fas-FasL interactions have been shown to play significant roles in immune system homeostasis. In this review the involvement of Fas and Fas ligand in cell death, with particular reference to the T cell, and the mechanism(s) by which they induce cell death is described. The role of AICD in immune system homeostasis and the controversy surrounding the role of FasL in immune privilege, inflammation, and so-called tumour counterattack is also discussed.