Experimental platforms to study blast injury.

Injuries sustained due to attacks from explosive weapons are multiple in number, complex in nature, and not well characterised. Blast may cause damage to the human body by the direct effect of overpressure, penetration by highly energised fragments, and blunt trauma by violent displacements of the body. The ability to reproduce the injuries of such insults in a well-controlled fashion is essential in order to understand fully the unique mechanism by which they occur, and design better treatment and protection strategies to alleviate the resulting poor long-term outcomes. This paper reports a range of experimental platforms that have been developed for different blast injury models, their working mechanism, and main applications. These platforms include the shock tube, split-Hopkinson bars, the gas gun, drop towers and bespoke underbody blast simulators.

Anabolic androgenic steroids, an easily forgotten cause of polycythaemia and cerebral infarction.

Excessive anabolic androgenic steroids (both exogenous and endogenous) are known causes of polycythaemia and ischaemic cardiovascular events. Despite this, they are commonly forgotten in the workup of patients. We report a case of exogenous anabolic androgenic steroid-induced polycythaemia and stroke and explore possible pitfalls for clinicians.

The SMAC mimetic, LCL-161, reduces survival in aggressive MYC-driven lymphoma while promoting susceptibility to endotoxic shock.

Inhibitor of apoptosis proteins (IAPs) antagonize caspase activation and regulate death receptor signaling cascades. LCL-161 is a small molecule second mitochondrial activator of caspase (SMAC) mimetic, which both disengages IAPs from caspases and induces proteasomal degradation of cIAP-1 and -2, resulting in altered signaling through the NFκB pathway, enhanced TNF production and sensitization to apoptosis mediated by the extrinsic pathway. SMAC mimetics are undergoing clinical evaluation in a range of hematological malignancies. Burkitt-like lymphomas are hallmarked by a low apoptotic threshold, conveying sensitivity to a range of apoptosis-inducing stimuli. While evaluating LCL-161 in the Eµ-Myc model of aggressive Burkitt-like lymphoma, we noted unexpected resistance to apoptosis induction despite 'on-target' IAP degradation and NFκB activation. Moreover, LCL-161 treatment of lymphoma-bearing mice resulted in apparent disease acceleration concurrent to augmented inflammatory cytokine-release in the same animals. Indiscriminate exposure of lymphoma patients to SMAC mimetics may therefore be detrimental due to both unanticipated prolymphoma effects and increased susceptibility to endotoxic shock.

Modified nasal dermoplasty technique for treatment of recurrent polyposis: preliminary results.

OBJECTIVE: To present and evaluate the use of nasal dermoplasty for control of recurrent nasal polyps. STUDY DESIGN: Prospective case series. METHOD: The mucosa of the fovea ethmoidalis and the lamina papyracea was replaced by a split-thickness skin graft. The follow-up period ranged from 2 to 12 months. RESULTS: Five patients underwent nasal dermoplasty for recurrent nasal polyposis. In three cases, the graft uptake was successful. Post-operatively, four patients reported they were in better condition than at the same interval after their previous operation. Recurrence of polyps was noted in all patients but not in the grafted areas. CONCLUSION: In this study, there was a high prevalence of successful graft uptake following nasal dermoplasty. This technique may have potential for the control of recurrent nasal polyps. Although it is demanding and time-consuming, it may reduce the need for multiple operations. Further research is justified to establish its efficacy.

Persistent complement-dependent anti-AnWj in a lymphoproliferative disorder: a case study and review.

AnWj is a high-incidence antigen present on the red blood cells (RBCs) of greater than 99 percent of the general population. A 58-year-old man underwent autologous hematopoietic stem cell transplantation (HSCT) for stage IVa mantle cell lymphoma. This procedure was complicated by failure to engraft, necessitating ongoing support with blood components. After a 2-month period of uneventful transfusion support, the patient experienced increasingly severe reactions with fever and evidence of intravascular hemolysis, including hemoglobinuria. Testing revealed a complement-dependent anti-AnWj. Phenotyping confirmed the AnWj- phenotype. Anti-AnWj was persistent despite immunosuppression, including treatment with allogeneic HSCT. Of interest, the pathogenesis of the downregulation of the graft AnWj in this patient is unclear.

Mice lacking the transcription factor subunit Rel can clear an influenza infection and have functional anti-viral cytotoxic T cells but do not develop an optimal antibody response.

Rel, a haemopoietic cell-restricted member of the NF-kappaB/Rel family of transcription factors, has recently been shown to be important in the function of B and T lymphocytes. In an attempt to understand the role of this protein in the immune response, we examined the ability of Rel(-/-) mice to counter an influenza virus infection. Normal levels of virus-specific cytotoxic T cells induced in Rel(-/-) mice were able to clear virus from the lungs, albeit with somewhat delayed kinetics compared to normal mice. Rel(-/-) mice did, however, display a markedly reduced T cell proliferative response to the virus, and exhibited impaired local and systemic influenza virus-specific antibody responses. This defect was sufficient to result in an inability of vaccinated mice, but not of previously infected mice, to acquire antibody-dependent protective immunity to reinfection with the same virus. These findings establish that during the response to influenza virus, Rel function allows optimal development of humoral immunity, a role that apparently cannot be fulfilled by other NF-kappaB/Rel proteins.

Human monocytes maintained in culture acquire functional responsiveness to platelet-activating factor that is independent of increases in protein tyrosine phosphorylation.

1. Acute (day 0) stimulation with platelet-activating factor (PAF) did not elicit superoxide anion (O2-) generation from adherent monocytes. However, by day 2 of culture, PAF induced an increase in O2- generation that was inhibited by pretreatment with the PAF receptor antagonist WEB 2086. 2. The lack of effect of PAF on O2- generation was not due to the absence of receptors, as PAF stimulated an increase in tyrosine phosphorylation and intracellular calcium ([Ca2+]i) on both days 0 and 2 of culture. 3. Pretreatment with the protein tyrosine kinase inhibitor methyl 2,5-dihydroxycinnamate inhibited PAF-induced tyrosine phosphorylation; however, this inhibitor failed to inhibit PAF-induced O2- generation. In contrast, pretreatment with the protein kinase C inhibitor staurosporine had no effect on PAF-induced tyrosine phosphorylation, but did inhibit PAF-induced O2- generation. 4. These results indicate that monocytes maintained in culture acquire a functional response to PAF through a mechanism that appears to be independent of PAF receptor expression, coupling to increases in [Ca2+]i or tyrosine phosphorylation.

The Rel subunit of NF-kappaB-like transcription factors is a positive and negative regulator of macrophage gene expression: distinct roles for Rel in different macrophage populations.

The role of Rel in the monocyte/macrophage lineage was examined in mice with an inactivated c-rel gene. Although the frequency of monocytic cells was normal in Rel-/- mice, we show that Rel serves distinct roles in regulating gene expression and immune effector function in different mature macrophage populations. Stimulated Rel-/- resident peritoneal macrophages produced higher than normal levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), but tumour necrosis factor-alpha (TNF-alpha) production was not induced. Diminished cytotoxic activity exhibited by resident Rel-/- macrophages was consistent with reduced nitric oxide production resulting from impaired up-regulation of inducible nitric oxide synthase expression. While a similar altered pattern of IL-6 and TNF-alpha expression was observed in stimulated Rel-/- peritoneal effusion macrophages, cytotoxic activity, nitric oxide, GM-CSF and G-CSF production by these cells was normal. The alternate regulation of certain genes in the two macrophage populations coincided with different patterns of nuclear Rel/NF-kappaB complexes expressed in normal resident and elicited cells. Collectively, these results establish that Rel is a positive or negative regulator of transcription in macrophages and that Rel has distinct roles in different macrophage populations.

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Mitogenic actions of endothelin-1 and epidermal growth factor in cultured airway smooth muscle.

1. Hyperplasia of airway smooth muscle contributes to the increase in bronchomotor responsiveness that characterizes asthma. We have investigated the mitogenic potential of endothelin-1 (ET-1) and epidermal growth factor (EGF) in guinea-pig cultured airway smooth muscle and the relationship of these actions to tyrosine phosphorylation and increases in intracellular calcium (Ca2+i). 2. ET-1 stimulated DNA and protein synthesis and also increased cell number, but these mitogenic actions were small compared with those of EGF. 3. ET-1 and EGF increased the level of tyrosine phophorylation of a range of proteins with apparent molecular weights between 80 and 100 kDa and also increased phosphorylation of a single protein of 33 kDa. 4. Ca2+i levels were increased by both ET-1 and EGF. However, concentrations of EGF three orders of magnitude higher than those having mitogenic actions or increasing protein tyrosine phosphorylation were required. ET-1 was a more potent stimulant of increases in intracellular calcium than of mitogenesis. 5. We conclude that elevation of Ca2+i is unlikely to be an important signal for the mitogenic action of EGF. It is suggested that stimulants at the EGF receptor (EGF and transforming growth factor alpha) may play a role in the airway smooth muscle hyperplasia in asthma.

Physiological and pathophysiological roles of nitric oxide.

Nitric oxide (NO), identified as the biochemical messenger of endothelial-dependent relaxation, is of obvious chemical simplicity, but the range and complexity of its biological actions are only now emerging. NO is an important determinant of vascular resistance, it reduces thrombogenicity of the vascular endothelium, contributes to non-specific, host-defence mechanisms, and is a neurotransmitter in the peripheral and central nervous systems. In addition to these physiological roles, there is now convincing evidence that excessive, prolonged production of NO contributes to tissue damage in septicemia, ischemia/reperfusion injury, and other inflammatory conditions.

Albumin inhibits platelet-activating factor (PAF)-induced responses in platelets and macrophages: implications for the biologically active form of PAF.

1. Platelet-activating factor (PAF) binds with high affinity to albumin leading Clay et al. (1990) to suggest that the active form of PAF is the albumin-PAF complex. 2. In the present study the proposal that albumin-bound, rather than monomeric PAF, is the active form of PAF at PAF receptors was critically evaluated by examining the effect of albumin on the potency of PAF in isolated platelets and macrophages. 3. Bovine serum albumin inhibited concentration-dependently PAF-induced responses in platelets and macrophages. The most probable explanation of this finding is that BSA reduced the concentration of free PAF. 4. Thus, we conclude that free PAF, rather than the albumin-PAF complex is the active form. Consequently, local concentrations of albumin will influence profoundly the potency of endogenously released PAF. Moreover, estimates of the affinity of PAF for PAF receptors made in buffers containing BSA, underestimate the true affinity of PAF for its receptors by approximately 3 orders of magnitude.

Structure-activity relationships for platelet-activating factor (PAF) and analogues reveal differences between PAF receptors on platelets and macrophages.

Analogues of PAF were examined for their potency in stimulating either platelet aggregation or macrophage superoxide anion generation. Modification of either the alkyl side-chain or the acetyl side-chain increased the relative potency of PAF analogues in macrophages, but all these compounds were more active in platelets. However, an analogue of PAF with an increased inter-ionic distance in the polar head group, hexanolamine PAF, showed a greater potency in macrophages than platelets. The latter compound also appeared to act as a partial agonist in both rabbit platelets and guinea-pig macrophages, but not in guinea-pig platelets. Differences in the rank order of potency of the PAF analogues in stimulating these cell elements suggest that platelet and macrophage PAF receptors differ.

1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho (N,N,N trimethyl) hexanolamine: an analogue of platelet-activating factor with partial agonist activity.

1. During studies of the role of platelet-activating factor (PAF) in macrophage superoxide anion generation (O2(-1], we identified an agonist action of the putative PAF receptor antagonist 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho (N,N,N-trimethyl) hexanolamine (hexanolamine PAF) in guinea-pig macrophages. The 1-O-octadecyl form of this compound has specific antagonist actions at PAF receptors. 2. The agonist properties of hexanolamine PAF were examined in rabbit washed platelets (aggregation) and in guinea-pig peritoneal macrophages (O2- generation). 3. Hexanolamine PAF induced significant platelet aggregation (50% of the PAF maximum). However, the omission of bovine serum albumin (BSA) from the Tyrode buffer resulted in a diminution of the response of washed platelets during storage for 24 h at 4 degrees C (7% of PAF maximum), whereas the maximum response to PAF was unaffected by storage for this period, irrespective of the presence of BSA. 4. Platelet aggregation induced by hexanolamine PAF was not accompanied by a detectable increase in intracellular calcium [Ca2+]i, whereas the aggregation response to PAF was preceded by a large rise in [Ca2+]i. 5. Hexanolamine PAF induced O2- generation in adherent macrophages, with a maximum response 45% of that to PAF. Hexanolamine PAF (100 nM), at a concentration equi-effective with PAF (1 nM) for stimulation of O2- generation in macrophages, induced an increase in [Ca2+]i which was significantly less than that induced by PAF. 6. PAF concentration-response curves were constructed in platelets or macrophages following pretreatment with hexanolamine PAF (0.1 and 1 microM). The interaction between PAF and the putative partial agonist (hexanolamine PAF) had the characteristics expected of a partial agonist interacting with a full agonist.7. Platelet aggregation induced by hexanolamine PAF was antagonized non-competitively by the PAF receptor antagonist, WEB 2086, whereas antagonism of PAF-induced aggregation by WEB 2086 was competitive. Macrophage 2- generation induced by hexanolamine PAF or PAF was antagonized by WEB 2086.8. These data indicate that hexanolamine PAF is a partial agonist at PAF receptors in macrophages and platelets. The inability of hexanolamine PAF to increase [Ca2+]i in platelets suggests that PAF receptors may be coupled to platelet aggregation by both Ca2 +-dependent and -independent pathways.