David D. Haines

Incidence of lung, eye, and skin lesions as late complications in 34,000 Iranians with wartime exposure to mustard agent.

Learning ObjectivesRecall the frequency and severity of chronic pulmonary, ocular, and cutaneous lesions in this study of 34,000 Iranians exposed in war to mustard agent and followed up after 13 to 20 years.Relate the physical and chemical properties of mustard agents to their biological effects in exposed persons.Describe the chronic clinical sequelae of mustard exposure as observed in the lungs, eyes, and skin.Describe a categorizing method for determining the severity of lung, eye, and skin lesions based on clinical criteria. Approximately 34,000 Iranians known to have sustained mustard agent exposure during the Iran–Iraq war of 1980–1988 and survived over a decade afterwards were screened for distribution of the most commonly occurring medical problems. In order of greatest incidence, these include lesions of the lungs (42.5%), eyes (39.3%), and skin (24.5%). Within each subpopulation, patients were ranked according to severity of lesions. Twenty-three percent to 37% of patients exhibited at least mild coverage, with 1.5% to 4.5% classed as moderate, and a much smaller population (0.023–1.0%) of the 34,000 patients exhibiting extensive (severe) lesional coverage. These results provide a comprehensive overview of the medical problem most common among mustard victims and could serve as a predictor of the likely impact of these weapons on health status of populations exposed to them during ongoing military conflicts.

Role of haeme oxygenase-1 in resolution of oxidative stress-related pathologies: focus on cardiovascular, lung, neurological and kidney disorders

The present review examines the role of the cytoprotective enzyme haeme oxygenase‐1 (HO‐1) in adaptive responses to inflammatory disease and explores strategies for its clinical use, with particular emphasis on use of therapeutic use of the enzyme using phytochemical inducers of HO‐1 such as extracts of Ginkgo biloba, curcumin, and flavonoids extracted from seeds of the sour cherry (Prunus cerasus). This laboratory has identified strategies by which combinations of dietary phytochemicals may be configured to synergistically strengthen immunoregulatory mechanisms that normally prevent inflammation from leading to disease. A major focus of this research initiative has been HO‐1, which is capable of substantially reducing oxidative stress by several mechanisms. HO‐1 metabolizes haeme that accumulates in tissues because of red blood cell turnover. Two products of this degradation – carbon monoxide (CO) and bilirubin – have potent capacity for reducing oxidative stress and for counteracting its effects. A description will be provided of how HO‐1 products maintain healthy tissue function and remediate oxidative tissue damage. This will be explored in four major organ systems, including the cardiovascular system, the lungs, the central nervous system and the kidneys. Particular focus will be given to the physiological coordination of cardiovascular functions mediated by CO produced by HO‐1 and to nitric oxide (NO), a gaseous second messenger expressed by nitric oxide synthetase. A major unifying theme of the present review is an exploration of the potential use of dietary phytochemical formulations as tools for the clinical application of HO‐1 in therapeutic reduction of oxidative stressors, with resultant improved treatment of inflammatory pathologies.

Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts

Effects of the calcineurin inhibitor FK506, the platelet-activating factor (PAF) antagonist, and free radical scavenger Ginkgo biloba extract, EGb 761, and their combination on reperfusion-induced ventricular fibrillation (VF), ventricular tachycardia (VT), and recovery of cardiac function were studied after 30 min of global ischemia followed by 2 h of reperfusion in isolated rat hearts. In the first series of studies, rats received a daily (oral) dose of 0, 1, 5, 10, 20, or 40 mg/kg/day FK506 for 10 days. FK506 dose-dependently reduced the incidence of reperfusion-induced total (irreversible plus reversible) VF from a value of 92% for untreated animals to 92% (NS), 83% (NS), 67% (NS), 33% (p<0.05), and 25% (p<0.05), for doses of 1-40 mg/kg/day, respectively, with effects on incidence of VT showing the same pattern. FK506, between 20 and 40 mg/kg/day, also resulted in significant recovery of postischemic cardiac function. In the second series of studies, rats were treated with EGb 761 alone or in combination with FK506. Whereas no significant reduction in arrhythmias or improvement in cardiac function resulted from a single intervention of EGb 761 at 25 mg/kg/day, combined treatment of rats with 25 mg/kg/day of EGb 761 and 1 or 5 mg/kg/day of FK506 resulted in a reduction in total and irreversible VF of 92% and 92% to 42% (p<0.05) and 33% (p<0.05), 25% (p<0.05) and 8% (p<0.05), respectively, versus untreated control animals, paralleled by similar effects on the incidence of VT and accompanied by significant improvements in postischemic cardiac function. Our results demonstrate a novel cardioprotective characteristic of FK506 and suggest that combination therapy by using FK506 plus EGb 761 synergistically improves postischemic cardiac function, while reducing the incidence of reperfusion-induced VF and VT, which may expand the clinical utility of FK506 and allow therapy with FK506 at lower doses than are currently useful.

MUSTARD GAS EXPOSURE AND CARCINOGENESIS OF LUNG

UNLABELLED Sulfur mustard (SM), also known as mustard gas, is an alkylating compound used as a chemical weapon in World War I and by Iraqi forces against Iranians and indigenous Iraqi Kurds during the Iran-Iraq War of the 1980s. Although SM is a proven carcinogen there are conflicting views regarding the carcinogenicity of a single exposure. The present study characterizes lung cancers formed in mustard gas victims from the Iran-Iraq War. METHODS AND MATERIALS Demographic information and tumor specimens were collected from 20 Iranian male lung cancer patients with single high-dose SM exposures during the Iran-Iraq War. Formalin-fixed, paraffin-embedded lung cancers were analyzed by immunohistochemistry for p53 protein. In addition, DNA was extracted from the tissues, PCR amplified and sequenced to identify mutations in the p53 and KRAS genes associated with SM exposure. RESULTS A relatively early age of lung cancer onset (ranging from 28 to 73 with a mean of 48) in mustard gas victims, particularly those in the non-smoking population (mean age of 40.7), may be an indication of a unique etiology for these cancers. Seven of the 20 patients developed lung cancer before the age of 40. Five of 16 cancers from which DNA sequence data was obtainable provided information on eight p53 mutations (within exons 5-8). These mutations were predominately G to A transitions; a mutation consistent with the DNA lesion caused by SM. Two of the lung cancers had multiple p53 point mutations, similar to results obtained from factory workers chronically exposed to mustard agent. No mutations were detected in the KRAS gene. DISCUSSION The distinguishing characteristics of lung carcinogenesis in these mustard gas victims suggest that a single exposure may increase the risk of lung cancer development in some individuals.

Management of multicellular senescence and oxidative stress

Progressively sophisticated understanding of cellular and molecular processes that contribute to age‐related physical deterioration is being gained from ongoing research into cancer, chronic inflammatory syndromes and other serious disorders that increase with age. Particularly valuable insight has resulted from characterization of how senescent cells affect the tissues in which they form in ways that decrease an organisms overall viability. Increasingly, the underlying pathophysiology of ageing is recognized as a consequence of oxidative damage. This leads to hyperactivity of cell growth pathways, prominently including mTOR (mammalian target of rapamycin), that contribute to a build‐up in cells of toxic aggregates such as progerin (a mutant nuclear cytoskeletal protein), lipofuscin and other cellular debris, triggering formation of senescent cellular phenotypes, which interact destructively with surrounding tissue. Indeed, senescent cell ablation dramatically inhibits physical deterioration in progeroid (age‐accelerated) mice. This review explores ways in which oxidative stress creates ageing‐associated cellular damage and triggers induction of the cell death/survival programs’ apoptosis, necrosis, autophagy and ‘necroapoptophagy’. The concept of ‘necroapoptophagy’ is presented here as a strategy for varying tissue oxidative stress intensity in ways that induce differential activation of death versus survival programs, resulting in enhanced and sustained representation of healthy functional cells. These strategies are discussed in the context of specialized mesenchymal stromal cells with the potential to synergize with telocytes in stabilizing engrafted progenitor cells, thereby extending periods of healthy life. Information and concepts are summarized in a hypothetical approach to suppressing whole‐organism senescence, with methods drawn from emerging understandings of ageing, gained from Cnidarians (jellyfish, corals and anemones) that undergo a unique form of cellular regeneration, potentially conferring open‐ended lifespans.

Pregnancy-Associated Changes in Peripheral Blood Lymphocyte Subpopulations in Normal Kuwaiti Women

It has recently been reported that healthy pregnancy is associated with systemic immunosuppression. The aim of this study was to evaluate the numbers and distribution of lymphocyte subpopulations in normal, healthy pregnant Kuwaiti women. Thirty-four healthy normotensive women in the 3rd trimester of pregnancy were studied using flow cytometry to define lymphocyte subpopulations and were compared with 16 non-pregnant women. A decrease in the absolute numbers of lymphocytes was observed affecting T cells (CD3+, CD4+, CD8+), B cells (CD19+), and natrural killer cells (CD16+/CD56+). When analyzed as a percentage of the total lymphocyte population, there was a significant decrease in B cells and an increase in CD4+ T cells. The T cell population revealed increased expression of CD25 on CD4+ and CD8+ cells, of HLA-DR on CD8+ cells, and of CD54 on CD4+ T cells. The reduced number of lymphocytes suggests that Kuwaiti females may be immunosuppressed in the 3rd trimester of pregnancy. The presence of activated CD4+ T cells could indicate the expression of a regulatory suppressor T cell population, as Treg cells are CD4+CD25+, and suppressor T cells are thought to be CD8+. Future work is required to explore the significance of these T cell populations in pregnancy.

Summative interaction between astaxanthin, Ginkgo biloba extract (EGb761) and vitamin C in suppression of respiratory inflammation: a comparison with ibuprofen.

In this study, combinations of Ginkgo biloba leaf extract (EGb761) plus the carotenoid antioxidant astaxanthin (ASX) and vitamin C were evaluated for a summative dose effect in the inhibition of asthma‐associated inflammation in asthmatic guinea‐pigs. Ovalbumin‐sensitized Hartley guinea‐pigs challenged with ovalbumin aerosol to induce asthma, were administered EGb761, ASX, vitamin C or ibuprofen. Following killing, bronchoalveolar lavage (BAL) fluid was evaluated for inflammatory cell infiltrates and lung tissue cyclic nucleotide content. Each parameter measured was significantly altered to a greater degree by drug combinations, than by each component acting independently. An optimal combination was identified that included astaxanthin (10 mg/kg), vitamin C (200 mg/kg) and EGb761 (10 mg/kg), resulting in counts of eosinophils and neutrophils each 1.6‐fold lower; macrophages 1.8‐fold lower, cAMP 1.4‐fold higher; and cGMP 2.04‐fold higher than levels in untreated, asthmatic animals (p < 0.05). In conclusion, EGb761, ASX and vitamin C are shown here to interact summatively to suppress inflammation with efficacy equal to or better than ibuprofen, a widely used non‐steroidal antiinflammatory drug (NSAID). Such combinations of non‐toxic phytochemicals constitute powerful tools for the prevention of onset of acute and chronic inflammatory disease if consumed regularly by healthy individuals; and may also augment the effectiveness of therapy for those with established illness. Copyright

Cardioprotection afforded by sour cherry seed kernel: the role of heme oxygenase-1

Abstract: Cardiovascular diseases are primary cause of death worldwide, particularly among populations with sedentary lifestyles and diets rich in animal products and processed foods. Currently, public health countermeasures to these disorders focus on costly and often marginally effective interventions administered only after the development of disease. These countermeasures are mainly palliative and fail to address the underlying causes of cardiac pathologies. Previously, the authors of this report have demonstrated that sour cherry seed kernel extract (SCSE), a nontoxic low-cost plant material, strongly preserves tissues through induction of heme oxygenase-1 (HO-1), a critical host antioxidant defense enzyme. This investigation seeks to characterize underlying mechanisms of SCSE-mediated tissue protection. Isolated hearts from Sprague–Dawley rats fed 30 mg·kg−1·d−1 SCSE for 8 weeks, and untreated controls were mounted in a “working heart” apparatus and subjected to ischemia and reperfusion. A panel of cardiac functional evaluations was conducted on each heart. Infarct size assessments were made along with Western blot and immunohistochemical analysis for selected proteins involved in cardiovascular homeostasis. SCSE treatment was observed to improve postischemic cardiac functions and suppress infarct size. Analysis of the outcomes produced by this study is consistent with SCSE cardioprotection that involve interaction of Bcl-2 and HO-1.

Sour cherry seed kernel extract increases heme oxygenase-1 expression and decreases representation of CD3+ TNF-α+ and CD3+IL-8+ subpopulations in peripheral blood leukocyte cultures from type 2 diabetes patients.

The present study evaluates a hypothesis that sour cherry (Prunus cerasus) seed extracts (SCE) modulate CD3+ T lymphocyte activity in ways predictive of potential for uses of SCE in management of inflammatory diseases. Peripheral blood mononuclear cells (PBMC) from 12 type 2 diabetes (T2DM) patients and eight healthy control subjects were cultured 24 h with 100 ng/ml lipopolysaccharide (LPS) to increase inflammatory signaling and co‐incubated with 0.5–100 µg/ml SCE. Cultures were evaluated by two‐color flow cytometry for percent representation of CD3+ IL8+ and CD3 + TNF‐α + cells which express interleukin‐8 (IL‐8), and tumor necrosis factor‐α, (TNF‐α+) respectively, and by enzyme‐linked immunoassay for lymphocyte‐associated heme oxygenase‐1 (HO‐1, known to be induced by SCE). SCE dosage ranges of 0.5–100 µg/ml in cell cultures significantly suppressed LPS‐increased CD3 + TNF‐α + and CD3 + IL8+ representation from all participants (p < 0.05), with greater pharmacological effect noted in suppression of CD3 + TNF‐α + noted in cells from T2DM patients versus healthy control subjects. These effects correlated with increased HO‐1 expression in SCE‐treated PBMC from all subjects (p < 0.05). Since TNF‐α and IL‐8 are diagnostic/prognostic biomarkers for many inflammatory syndromes, the capacity of SCE to down‐regulate representation of cells that express them suggests potential for therapeutic use of SCE in T2DM and other diseases. Copyright

Evaluation of Systemic and Dermal Toxicity and Dermal Photoprotection by Sour Cherry Kernels

The present report describes outcomes of animal studies conducted to determine the systemic and dermal toxicity of Prunus cerasus (sour cherry) seed kernel contents; and a separate evaluation of the photoprotective capacity of the kernel oil fraction. B6 mice and Hartley guinea‐pigs were used for these experiments. Dosage groups of 6–8 animals were administered whole kernel meal in a dose range of 0–3000 mg/kg by gavage for 8 days, following which they were killed. The liver and kidney weights were recorded and histological examination performed on sections of these organs. Kidney function was assessed as blood urea nitrogen and creatinine and liver function by measurement of serum glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and alkaline phosphatase.