Toll-like receptor 3 L412F polymorphism promotes a persistent clinical phenotype in pulmonary sarcoidosis.

BACKGROUND/INTRODUCTION: Sarcoidosis is a multi-systemic disorder of unknown etiology, characterized by the presence of non-caseating granulomas in target organs. In 90% of cases, there is thoracic involvement. Fifty to seventy percent of pulmonary sarcoidosis patients will experience acute, self-limiting disease. For the subgroup of patients who develop persistent disease, no targeted therapy is currently available. AIM: To investigate the potential of the single nucleotide polymorphism (SNP), Toll-like receptor 3 Leu412Phe (TLR3 L412F; rs3775291), as a causative factor in the development of and in disease persistence in pulmonary sarcoidosis. To investigate the functionality of TLR3 L412F in vitro in primary human lung fibroblasts from pulmonary sarcoidosis patients. DESIGN: SNP-genotyping and cellular assays, respectively, were used to investigate the role of TLR3 L412F in the development of persistent pulmonary sarcoidosis. METHODS: Cohorts of Irish sarcoidosis patients (n = 228), healthy Irish controls (n = 263) and a secondary cohort of American sarcoidosis patients (n = 123) were genotyped for TLR3 L412F. Additionally, the effect of TLR3 L412F in primary lung fibroblasts from pulmonary sarcoidosis patients was quantitated following TLR3 activation in the context of cytokine and type I interferon production, TLR3 expression and apoptotic- and fibroproliferative-responses. RESULTS: We report a significant association between TLR3 L412F and persistent clinical disease in two cohorts of Irish and American Caucasians with pulmonary sarcoidosis. Furthermore, activation of TLR3 in primary lung fibroblasts from 412 F-homozygous pulmonary sarcoidosis patients resulted in reduced IFN-ß and TLR3 expression, reduced apoptosis- and dysregulated fibroproliferative-responses compared with TLR3 wild-type patients. DISCUSSION/CONCLUSION: This study identifies defective TLR3 function as a previously unidentified factor in persistent clinical disease in pulmonary sarcoidosis and reveals TLR3 L412F as a candidate biomarker.

Changes in Nutritional and Functional Status in Longer Stay Patients Admitted to a Geriatric Evaluation and Management Unit.

OBJECTIVES: Malnutrition and functional decline are common in older inpatients admitted to subacute care settings. However the association between changes in nutritional status and relevant functional outcomes remains under-researched. This study examined changes in nutritional status, function and mobility in patients admitted to a Geriatric Evaluation and Management (GEM) unit who had a length of stay (LOS) longer than 21 days. DESIGN: A prospective, observational study. SETTING: Two GEM units at St Vincent's Hospital Melbourne, Australia. PARTICIPANTS: Patients admitted to the GEM units who stayed longer than 21 days were included in the study. MEASUREMENTS: Patients were assessed on admission and prior to discharge using the Subjective Global Assessment (SGA), Functional Independence Measure (FIM) motor domain and the Modified Elderly Mobility Scale (MEMS). RESULTS: Fifty-nine patients (Mean age 84.0 ± 7 years) met the required length of stay and were included in the study. Fifty-four per cent (n=32) were malnourished on admission (SGA B/C) and 44% (n=26) were malnourished on discharge. Twenty-two per cent (n=13) improved SGA category, 75% remained stable (n=44) and 3% deteriorated (n=2) from admission to discharge. Total Motor FIM scores significantly increased from admission to discharge in both the improved (p<0.001) and stable or deteriorated (p<0.001) nutritional status groups. Subjects who improved in nutritional status had a significantly higher MEMS score at discharge (p<0.001). CONCLUSION: On admission to the GEM unit, just over half the included patients were rated as malnourished defined by SGA category. Nearly one quarter of the sample had improved their nutritional status at the time of discharge. Improvement in nutritional status was associated with greater improvement in mobility scores. Further studies are required to investigate the effectiveness of nutrition interventions, which will inform models of care aiming to optimise nutritional, functional, and associated clinical outcomes in patients admitted to GEM units.

Novel strategies for targeting innate immune responses to influenza.

We previously reported that TLR4(-/-) mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist Eritoran blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post infection. Herein we extend these findings: anti-TLR4- or -TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h before PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4(-/-) mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks high-mobility group B1 (HMGB1)-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI; (iii) interleukin (IL)-1ß contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the antiviral drug oseltamivir were administered starting 4 days post infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.

The ontogeny of fairness in seven societies.

A sense of fairness plays a critical role in supporting human cooperation. Adult norms of fair resource sharing vary widely across societies, suggesting that culture shapes the acquisition of fairness behaviour during childhood. Here we examine how fairness behaviour develops in children from seven diverse societies, testing children from 4 to 15 years of age (n = 866 pairs) in a standardized resource decision task. We measured two key aspects of fairness decisions: disadvantageous inequity aversion (peer receives more than self) and advantageous inequity aversion (self receives more than a peer). We show that disadvantageous inequity aversion emerged across all populations by middle childhood. By contrast, advantageous inequity aversion was more variable, emerging in three populations and only later in development. We discuss these findings in relation to questions about the universality and cultural specificity of human fairness.

Recent insights into the role of Toll-like receptors in viral infection.

Toll-like receptors (TLRs) have a central role in innate immunity as they detect conserved pathogen-associated molecular patterns (PAMPs) on a range of microbes, including viruses, leading to innate immune activation and orchestration of the adaptive immune response. To date, a large number of viruses have been shown to trigger innate immunity via TLRs, suggesting that these receptors are likely to be important in the outcome to viral infection. This suggestion is supported by the observation that many viruses have evolved mechanisms not only to evade the innate immune system, but also to subvert it for the benefit of the virus. In this review we will discuss earlier evidence, mainly from knock-out mice studies, implicating TLRs in the innate immune response to viruses, in light of more recent clinical data demonstrating that TLRs are important for anti-viral immunity in humans.

An arms race: innate antiviral responses and counteracting viral strategies.

Viral recognition is mediated by different classes of PRRs (pattern-recognition receptors) among which the TLRs (Toll-like receptors) and the RLHs [RIG (retinoic-acid-inducible)-like helicases] play major roles. The detection of PAMPs (pathogen-associated molecular patterns) by these PRRs leads to the initiation of signalling pathways that ultimately result in the activation of transcription factors such as NF-kappaB (nuclear factor kappaB) and IRF-3 [IFN (interferon) regulatory factor-3] and IRF-7 and the induction of pro-inflammatory cytokines and type I IFNs. Viruses have evolved a fine-tuned mechanism to evade detection by the immune system or to interfere with the resulting signalling pathways. Here, we discuss viral evasion proteins that specifically interfere with TLR and/or RLH signalling.

Translational mini-review series on Toll-like receptors: recent advances in understanding the role of Toll-like receptors in anti-viral immunity.

(TLRs) respond to pathogens to initiate the innate immune response and direct adaptive immunity, and evidence to date suggests that they have a role in the detection of viruses. Many viral macromolecules have been shown to activate anti-viral signalling pathways via TLRs, leading to the induction of cytokines and interferons, while viruses also have means of not only evading detection by TLRs, but also of subverting these receptors for their own purposes. This review discusses the role of TLRs in the context of other known viral detection systems, and examines some of the often surprising results from studies using mice deficient in TLRs and their adaptors, in an attempt to unravel the particular contribution of TLRs to anti-viral immunity.

Evasion of innate immunity by vaccinia virus.

Vaccinia virus, a member of the Poxviridae, expresses many proteins involved in immune evasion. In this review, we present a brief characterisation of the virus and its effects on host cells and discuss representative secreted and intracellular proteins expressed by vaccinia virus that are involved in modulation of innate immunity. These proteins target different aspects of the innate response by binding cytokines and interferons, inhibiting cytokine synthesis, opposing apoptosis or interfering with different signalling pathways, including those triggered by interferons and toll-like receptors.

Integrated luminometer for the determination of trace metals in seawater using fluorescence, phosphorescence and chemiluminescence detection.

The paper describes an integrated luminometer able to perform fluorescence (FL), room temperature phosphorescence (RTP) and chemiluminescence (CL) measurements on seawater samples. The technical details of the instrumentation are presented together with flow injection (FI) manifolds for the determination of cadmium and zinc (by FL), lead (RTP) and cobalt (CL). The analytical figures of merit are given for each manifold and results are presented for the determination of the four trace metals in seawater reference materials (NASS-5, SLEW-2) and Scheldt estuarine water samples.

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Transactivation by the p65 subunit of NF-kappaB in response to interleukin-1 (IL-1) involves MyD88, IL-1 receptor-associated kinase 1, TRAF-6, and Rac1.

We have examined the involvement of components of the interleukin-1 (IL-1) signaling pathway in the transactivation of gene expression by the p65 subunit of NF-kappaB. Transient transfection of cells with plasmids encoding wild-type MyD88, IL-1 receptor-associated kinase 1 (IRAK-1), and TRAF-6 drove p65-mediated transactivation. In addition, dominant negative forms of MyD88, IRAK-1, and TRAF-6 inhibited the IL-1-induced response. In cells lacking MyD88 or IRAK-1, no effect of IL-1 was observed. Together, these results indicate that MyD88, IRAK-1, and TRAF-6 are important downstream regulators of IL-1-mediated p65 transactivation. We have previously shown that the low-molecular-weight G protein Rac1 is involved in this response. Constitutively active RacV12-mediated transactivation was not inhibited by dominant negative MyD88, while dominant negative RacN17 inhibited the MyD88-driven response, placing Rac1 downstream of MyD88 on this pathway. Dominant negative RacN17 inhibited wild-type IRAK-1- and TRAF-6-induced transactivation, and in turn, dominant negative IRAK-1 and TRAF-6 inhibited the RacV12-driven response, suggesting a mutual codependence of Rac1, IRAK-1, and TRAF-6 in regulating this pathway. Finally, Rac1 was found to associate with the receptor complex via interactions with both MyD88 and the IL-1 receptor accessory protein. A pathway emanating from MyD88 and involving IRAK-1, TRAF-6, and Rac1 is therefore involved in transactivation of gene expression by the p65 subunit of NF-kappaB in response to IL-1.

Vitamin C inhibits NF-kappa B activation by TNF via the activation of p38 mitogen-activated protein kinase.

The transcription factor NF-kappaB is a central mediator of altered gene expression during inflammation, and is implicated in a number of pathologies, including cancer, atherosclerosis, and viral infection. We report in this study that vitamin C inhibits the activation of NF-kappaB by multiple stimuli, including IL-1 and TNF in the endothelial cell line ECV304 and in primary HUVECs. The induction of a NF-kappaB-dependent gene, IL-8, by TNF was also inhibited. The effect requires millimolar concentrations of vitamin C, which occur intracellularly in vivo, particularly during inflammation. Vitamin C was not toxic to cells, did not inhibit another inducible transcription factor, STAT1, and had no effect on the DNA binding of NF-kappaB. Inhibition by vitamin C was not simply an antioxidant effect, because redox-insensitive pathways to NF-kappaB were also blocked. Vitamin C was shown to block IL-1- and TNF-mediated degradation and phosphorylation of I-kappaBalpha (inhibitory protein that dissociates from NF-kappaB), due to inhibition of I-kappaB kinase (IKK) activation. Inhibition of TNF-driven IKK activation was mediated by p38 mitogen-activated protein kinase, because treatment of cells with vitamin C led to a rapid and sustained activation of p38, and the specific p38 inhibitor SB203580 reversed the inhibitory effect of vitamin C on IKK activity, I-kappaBalpha phosphorylation, and NF-kappaB activation. The results identify p38 as an intracellular target for high dose vitamin C.

A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization.

Changes in iron supply to oceanic plankton are thought to have a significant effect on concentrations of atmospheric carbon dioxide by altering rates of carbon sequestration, a theory known as the 'iron hypothesis'. For this reason, it is important to understand the response of pelagic biota to increased iron supply. Here we report the results of a mesoscale iron fertilization experiment in the polar Southern Ocean, where the potential to sequester iron-elevated algal carbon is probably greatest. Increased iron supply led to elevated phytoplankton biomass and rates of photosynthesis in surface waters, causing a large drawdown of carbon dioxide and macronutrients, and elevated dimethyl sulphide levels after 13 days. This drawdown was mostly due to the proliferation of diatom stocks. But downward export of biogenic carbon was not increased. Moreover, satellite observations of this massive bloom 30 days later, suggest that a sufficient proportion of the added iron was retained in surface waters. Our findings demonstrate that iron supply controls phytoplankton growth and community composition during summer in these polar Southern Ocean waters, but the fate of algal carbon remains unknown and depends on the interplay between the processes controlling export, remineralisation and timescales of water mass subduction.

Importance of stirring in the development of an iron-fertilized phytoplankton bloom.

The growth of populations is known to be influenced by dispersal, which has often been described as purely diffusive. In the open ocean, however, the tendrils and filaments of phytoplankton populations provide evidence for dispersal by stirring. Despite the apparent importance of horizontal stirring for plankton ecology, this process remains poorly characterized. Here we investigate the development of a discrete phytoplankton bloom, which was initiated by the iron fertilization of a patch of water (7 km in diameter) in the Southern Ocean. Satellite images show a striking, 150-km-long bloom near the experimental site, six weeks after the initial fertilization. We argue that the ribbon-like bloom was produced from the fertilized patch through stirring, growth and diffusion, and we derive an estimate of the stirring rate. In this case, stirring acts as an important control on bloom development, mixing phytoplankton and iron out of the patch, but also entraining silicate. This may have prevented the onset of silicate limitation, and so allowed the bloom to continue for as long as there was sufficient iron. Stirring in the ocean is likely to be variable, so blooms that are initially similar may develop very differently.

A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling.

Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll/IL-1 receptor (TIR) domain, a motif that defines the IL-1/Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NFkappaB, and A52R potently blocked both IL-1- and TLR4-mediated NFkappaB activation. MyD88 is a TIR domain-containing adapter molecule known to have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R are likely to represent a mechanism used by vaccinia virus of suppressing TIR domain-dependent intracellular signaling.

The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products.

The interleukin-1 (IL-1) receptor/Toll-like receptor (TLR) superfamily is a recently defined and expanding group of receptors that participate in host responses to injury and infection. The superfamily is defined by the Toll/IL-1 receptor (TIR) domain, which occurs in the cytosolic region of family members, and is further subdivided into two groups based on homology to either the Type I IL-1 receptor or Drosophila Toll receptor extracellular domain. The former group includes the receptor for the important Th1 cytokine IL-18, and T1/ST2, which may have a role in Th2 cell function. The latter group includes six mammalian TLRs, including TLR2 and TLR4, that largely mediate the host response to gram-positive and gram-negative bacteria, respectively. Whether bacterial products are actual ligands for TLRs, or whether they generate ligands via as yet unidentified pattern recognition receptors, has yet to be determined. Signaling pathways activated via the TIR domain trigger the activation of downstream kinases, and transcription factors such as NF-kappaB, and involve the adaptor protein MyD88, which itself contains a TIR domain.

Ras, protein kinase C zeta, and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation of NF-kappa B by hyaluronic acid fragments in T-24 carcinoma cells.

We have investigated the ability of hyaluronic acid (HA) fragments to activate the transcription factor NF-kappa B. HA fragments activated NF-kappa B in the cell lines T-24, HeLa, MCF7, and J774. Further studies in T-24 cells demonstrated that HA fragments also induced I kappa B alpha phosphorylation and degradation, kappa B-linked reporter gene expression, and ICAM-1 promoter activity in an NF-kappa B-dependent manner. The effect of HA was size dependent as neither disaccharide nor native HA were active. CD44, the principal cellular receptor for HA, was critical for the response because the anti-CD44 Ab IM7.8.1 blocked the effect on NF-kappa B. HA fragments activated the I kappa B kinase complex, and the effect on a kappa B-linked reporter gene was blocked in T-24 cells expressing dominant negative I kappa B kinases 1 or 2. Activation of protein kinase C (PKC) was required because calphostin C inhibited NF-kappa B activation and I kappa B alpha phosphorylation. In particular, PKC zeta was required because transfection of cells with dominant negative PKC zeta blocked the effect of HA fragments on kappa B-linked gene expression and HA fragments increased PKC zeta activity. Furthermore, damnacanthal and manumycin A, two mechanistically distinct inhibitors of Ras, blocked NF-kappa B activation. Transfection of T-24 cells with dominant negative Ras (RasN17) blocked HA fragment-induced kappa B-linked reporter gene expression, and HA fragments activated Ras activity within 5 min. Taken together, these studies establish a novel signal transduction cascade emanating from CD44 to Ras, PKC zeta, and I kappa B kinase 1 and 2.

Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries.

Nuclear factor-kappaB (NFKB) is a transcription factor with a pivotal role in inducing genes involved in physiological processes as well as in the response to injury and infection. A model has been proposed whereby the diverse agents that activate NFkappaB do so by increasing oxidative stress within the cell. Activation of NFkappaB involves the phosphorylation and subsequent degradation of an inhibitory protein, IKB, and recently many of the proximal kinases and adaptor molecules involved in this process have been elucidated. Additionally, we now understand in detail the NFkappaB activation pathway from cell membrane to nucleus for interleukin-1 (IL-1) and tumour necrosis factor (TNF). This review revisits the evidence for the oxidative stress model in light of these recent findings, and finds little in the new information to rationalise or justify a central role for oxidative stress in NF-kappaB activation. We demonstrate that much of the evidence for the involvement of oxidative stress is either specific to a stimulus in a particular cell line or open to reinterpretation. In particular, the activation of NFkappaB by hydrogen peroxide is cell-specific and distinct from physiological activators such as IL-1 and TNF, while inhibition by antioxidants, also found to be cell- and stimulus-specific, can involve diverse and unexpected targets which may be distinct from redox modulation. We conclude that in most cases the role of oxidative stress in NF-kappaB activation is at best facilitatory rather than causal, if a role exists at all. In addition, other evidence suggests a role for lipid peroxides in pathways where such a role exists. In future, when a role for oxidative stress in a pathway is postulated, the challenge will be to show which particular kinases or adaptor molecules, if any, are redox-modulated.

The production of a reactive oxygen intermediate during the induction of apoptosis by cytotoxic insult.

We report the protective effects of two novel antioxidant compounds, 3 beta-doxyl-5 alpha cholestane and 2,2,6,6-tetramethyl-piperdinoxyl, in HL-60 and U937 leukemic cells subjected to a number of cytotoxic insults. In addition, the rapid production of peroxide is demonstrated as a general response to cytotoxic agents in these leukemic cell lines, indicating that changes in the redox status of a leukemic cell may contribute to the ultimate death of these cells. Closer examination of this peroxide production has identified enzymic production and/or disruption of resident antioxidants as possible sources. However, in contrast to recent reports from other model systems, mitochondrial transmembrane depolarization did not appear to be required for the production of peroxide in these cells. Finally, we demonstrated the activation of the redox-sensitive transcription factor, NF-kappa B, in response to these cytotoxic insults.

Lipid peroxidation is involved in the activation of NF-kappaB by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304. Lack of involvement of H2O2 in NF-kappaB activation by either cytokine in both primary and transformed endothelial cells.

It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-kappaB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-kappaB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-kappaB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-kappaB and stimulation of IkappaBalpha degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive to N-acetyl-L-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-kappaB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-kappaB activation and IkappaBalpha degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6, 6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-kappaB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-kappaB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-kappaB activation by tumor necrosis factor but not interleukin-1. Taken together, the results argue against a role for H2O2 in NF-kappaB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-kappaB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation.