Future directions for the discovery of natural product-derived immunomodulating drugs: an IUPHAR positional review.

Drug discovery from natural sources is going through a renaissance, having spent many decades in the shadow of synthetic molecule drug discovery, despite the fact that natural product-derived compounds occupy a much greater chemical space than those created through synthetic chemistry methods. With this new era comes new possibilities, not least the novel targets that have emerged in recent times and the development of state-of-the-art technologies that can be applied to drug discovery from natural sources. Although progress has been made with some immunomodulating drugs, there remains a pressing need for new agents that can be used to treat the wide variety of conditions that arise from disruption, or over-activation, of the immune system; natural products may therefore be key in filling this gap. Recognising that, at present, there is no authoritative article that details the current state-of-the-art of the immunomodulatory activity of natural products, this in-depth review has arisen from a joint effort between the International Union of Basic and Clinical Pharmacology (IUPHAR) Natural Products and Immunopharmacology Sections, with contributions from a number of world-leading researchers in the field of natural product drug discovery, to provide a "position statement" on what natural products has to offer in the search for new immunomodulatory argents. To this end, we provide a historical look at previous discoveries of naturally occurring immunomodulators, present a picture of the current status of the field and provide insight into the future opportunities and challenges for the discovery of new drugs to treat immune-related diseases.

The Ethnopharmacologic Contribution to Bioprospecting Natural Products.

Descriptions of the use of natural products in traditional medicine have served as starting points for new therapeutics. The details of the traditional use of these organisms can provide important information for future drug discovery and development efforts. Recent technologic advances provide the framework to leverage ethnopharmacologic data in the drug discovery process. Information on the traditional harvest, preparation, storage, and administration of the organisms, and the natural products they contain, provides valuable details regarding characteristics of the active compounds. Importantly, researchers can now rapidly analyze and identify the multiple, and often synergistic, compounds contained in these natural products. Although we are entering the acme of ethnopharmacology, where information regarding the traditional use of organisms can provide valuable natural product leads and accelerate the identification of new therapeutics, this ethnopharmacologic resource is threatened by the loss of traditional medicine knowledge and extinction of organisms.

Apoptosis of hippocampal pyramidal neurons is virus independent in a mouse model of acute neurovirulent picornavirus infection.

Many viruses, including picornaviruses, have the potential to infect the central nervous system (CNS) and stimulate a neuroinflammatory immune response, especially in infants and young children. Cognitive deficits associated with CNS picornavirus infection result from injury and death of neurons that may occur due to direct viral infection or during the immune responses to virus in the brain. Previous studies have concluded that apoptosis of hippocampal neurons during picornavirus infection is a cell-autonomous event triggered by direct neuronal infection. However, these studies assessed neuron death at time points late in infection and during infections that lead to either death of the host or persistent viral infection. In contrast, many neurovirulent picornavirus infections are acute and transient, with rapid clearance of virus from the host. We provide evidence of hippocampal pathology in mice acutely infected with the Theiler's murine encephalomyelitis picornavirus. We found that CA1 pyramidal neurons exhibited several hallmarks of apoptotic death, including caspase-3 activation, DNA fragmentation, and chromatin condensation within 72 hours of infection. Critically, we also found that many of the CA1 pyramidal neurons undergoing apoptosis were not infected with virus, indicating that neuronal cell death during acute picornavirus infection of the CNS occurs in a non-cell-autonomous manner. These observations suggest that therapeutic strategies other than antiviral interventions may be useful for neuroprotection during acute CNS picornavirus infection.

Aloin induces apoptosis in Jurkat cells.

Aloe is widely used as a dietary supplement. However, there are continuing concerns over the toxicity and the purity of aloe-based products. The primary class of compounds responsible for aloe-induced toxicity are anthraquinones. One of these, aloe-emodin, has been extensively investigated for apoptosis inducing effects. Conversely, the precursor to aloe-emodin, aloin, has been subjected to only minimal investigation of any cytotoxic effects. Jurkat T cells, an established model for the study of compound toxicity, were used to evaluate the effect of aloin on cell viability. Cells were analyzed using flow cytometry and microscopy for cell size and granularity, cell membrane integrity, mitochondrial membrane potential, and cell cycle profile. Treatment with aloin resulted in a reduction in cell size, compromised membrane integrity, and loss of mitochondrial membrane potential in a dose-dependent manner. Additionally, treatment with aloin resulted in alteration of the cell cycle, specifically a block at G2/M phase. Importantly, the loss of cell membrane integrity was preceded by a loss of mitochondrial membrane potential, suggesting a mitochondrial-dependent pathway for aloin-induced apoptosis. These observations provide insight into the potential mechanisms of aloin-induced toxicity and thus, perhaps, aloe preparation-induced toxicity. Furthermore, because of the concern over the safety of aloe-based supplements, this work suggests that aloe supplements not containing aloin may be safer than aloe supplements containing aloin, and that aloin should be considered in addition to concentrations of aloe-emodin.

Chronic Lead Intoxication From Eating Wild-Harvested Game.

BACKGROUND: The purpose of this article is to determine if conversion from eating wild game harvested with lead-based ammunition to nonlead-based ammunition results in lower blood lead levels. Supersonic injection of toxin-leeching frangible projectiles into food is intuitively bad. As much as 95% of the ~13.7 million hunters in the United States choose shrapnel-inducing lead bullets to kill game; in addition, not harvesting meat is an incarcerable crime. A lead ammunition ban on certain federal lands was recently rescinded and the National Rifle Association refutes any risk from eating lead bullet-harvested game. METHODS: A patient subsisting solely on lead-shot meat was converted to non-lead ammunition and his blood lead level tracked. Concomitant with his conversion to nonlead ammunition, a controlled experiment was performed using the patient's bullets to determine his daily lead intake from lead-shot meat. RESULTS: While eating lead-shot meat, the patient was consuming 259.3 ± 235.6 µg of lead daily and his blood lead level was 74.7 µg/dL. Conversion to nonlead ammunition was associated with a reduced blood lead level. CONCLUSIONS: Unsafe blood lead levels can occur from eating game harvested with lead ammunition. Physicians should warn hunting patients of this potential risk and counsel them about the availability of nonlead ammunition alternatives.

Picornaviruses and cell death.

Members of the picornavirus family, including poliovirus and foot-and-mouth disease virus, are widespread pathogens of humans and domestic animals. Recent global developments in the resurgence of poliovirus infection and in the control of foot-and-mouth disease infection highlight the problems caused by the ability of picornaviruses to alter the apoptotic machinery of host cells and establish persistent infections. Despite the medical, economic and social impact of this family of viruses, little information exists that integrates the mechanisms of cell death and damage induced by related family members. Fortunately, examination of the reported roles and functions of individual viral proteins from multiple picornaviruses makes it possible to surmise canonical functions for these proteins. This review analyzes the canonical function of picornavirus proteins involved in the alteration of apoptotic homeostasis in infected host cells.

Beta-methylamino-alanine (BMAA) injures hippocampal neurons in vivo.

The unusually high incidence of amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC) among the Chamorro people of Guam has fueled an intense search for the etiologic agent responsible for this neurodegenerative disease. Recently, a biomagnification hypothesis was proposed to account for the role of dietary consumption of beta-methylamino-alanine (BMAA) in patients with ALS/PDC. However, this hypothesis is hotly debated and a direct association between BMAA and neuronal injury in vivo has been lacking. We provide evidence that introduction of BMAA into the CNS of mice leads to sporadic death of hippocampal neurons, supporting a direct causal link between BMAA and neuronal injury.

Mitochondrial involvement in Atuna racemosa induced toxicity.

Our group has developed a system to extract information regarding potential novel pharmaceuticals from historic herbal texts. We have shown that one of the plants identified through this technique has the purported antibacterial properties suggested by the text. Here, the toxicity of this antibacterial extract was examined. Using a Jurkat cell model, a therapeutic window between the minimal inhibitory concentration for Gram-positive bacteria and the dose-dependent toxicity of the Atuna racemosa extract was established. Using cells with a mutated caspase 8, it was shown that the toxicity does not involve caspase 8. However, by transmission electron microscopy and a potentiometric dye, the toxicity was shown to involve the mitochondria. This toxicity also resulted in DNA cleavage and activation of caspase 3. This work suggests that the extract, originally reported as an antimicrobial therapeutic in a 400-year-old Dutch herbal text, may maintain a therapeutic window as an antibiotic. Furthermore, this work shows toxicity would occur in a mitochondrial dependent fashion.

Hold on to your memories: how recent findings will impact plant-based drug discovery.

It is accepted that genetic traits favoring the survival and reproduction of individual organisms are more successful and more likely to be passed on, and that this process of natural selection underlies the broader adaptations of species. However, the demonstration that an individual plant is able to impart 'untested' transgenerational changes to its offspring challenges this paradigm, and indicates an injection point for novelty into the overall adaptation process of plants. Understanding the basis of these newly discovered variations in metabolite expression will have broad implications for the field of ethnobotany. This discipline is predicated on knowledge garnered from generations of trial and error experimentation to identify medicinal properties in plants. Thus, the discovery of novel pathways to alter metabolite profiles in plant progeny also alter a fundamental assumption of the discipline: that plants with medicinal properties a thousand years ago will still have medicinal properties today.

Antibacterial properties and toxicity of Atuna racemosa extract depend on kernel maturity.

Through data mining a historic herbal text, we identified Atuna racemosa-Raf. as a plant with alleged antibacterial properties. We have shown that these purported antibacterial properties are most prominent in the kernel of the nut of the plant. While working with traditional healers in Samoa during a botanical collection trip, we identified a range of maturity stages of the kernel. Here we show that the antibacterial properties are different at different stages of kernel maturity, and that the immature kernels have a lower minimal inhibitory concentration (MIC) than the mature kernels. Additionally, we show there is a negative correlation between the antibacterial properties and cytotoxic properties (a stronger antibiotic is less cytotoxic), suggesting there are two separate compounds with disparate characteristics. These findings have implications for the use of this natural product as an antibiotic and chemotherapeutic agent.

A randomized Phase I study of Atuna racemosa: a potential new anti-MRSA natural product extract.

AIM: We previously reported significant antimicrobial activity against Gram-positive bacteria from the extract of the Atun tree (Atuna racemosa), identified through rapid digital bioprospecting of a 400-year-old historic herbal text. Toxicity studies in human cell lines showing safety, combined with the ethnomedical descriptions of botanical use, suggested that this extract might be clinically useful against topical Gram-positive bacteria infections. MATERIALS AND METHODS: Using a minimal inhibitory concentration assay, we examined the susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) to an extract of the kernel of the Atun tree (Atuna racemosa). Additionally, a maximum tolerated topical application of the extract was determined in a randomized, double-blind, placebo-controlled pilot clinical trial. RESULTS: Here we report that the effectiveness of this Atuna racemosa extract against MRSA (MIC=16-32microg/mL) is on par with currently available last-line antibiotics, while it remains well tolerated in short-term topical applications of 10 times the minimally inhibitory concentration. CONCLUSIONS: Although further studies are needed to determine safety and clinical efficacy, this effective extract, identified in a 400-year-old historic herbal text, may prove to be clinically useful in the treatment of MRSA.

Cytotoxic properties of Diospyros seychellarum extract.

During a recent botanical expedition in the Seychelles archipelago we identified healers using Diospyros seychellarum as a tonic. Since this plant lacks any medicinal record in the current literature, we assessed the cytotoxic potential of D. seychellarum. Using Jurkat cells as a model system we show, by flow cytometry, that treatment with the leaf extract results in mitochondrial depolarization and subsequent loss of cellular membrane integrity. Additionally, by transmission electron microscopy, we show that treatment with the extract results in chromatin condensation, mitochondrial swelling, and loss of nuclear membrane integrity. Through these morphological and biochemical observations we concluded that the extract of Diospyros seychellarum is able to induce apoptosis. While it is difficult to extrapolate a potential pharmacologic function based on the ethnomedical use as a tonic, the ability of this extract to induce apoptosis warrants further investigation of the medicinal properties of this plant.

Potential role of high-stress employment in hypertension.

We report a patient with a reduction in blood pressure through cessation of high-stress employment.

A prospective observational study assessing the feasibility of measuring blood lead levels in New Zealand hunters eating meat harvested with lead projectiles.

There is no safe level of lead exposure. Correlations suggest that hunters harvesting wild game with lead bullets may be at risk of lead exposure through eating minute lead particles from shrapnel in their wild game. This feasibility study will determine if it is possible to conduct an interventional controlled, blinded study to evaluate if there is a causal relationship between meat harvested with lead bullets and elevated blood lead levels in those who consume the meat. This is an observational case crossover study and the primary outcome is blood lead levels. Individuals will have blood lead levels measured 2-4 days after eating one serving of meat harvested with lead bullets. At three potential washout periods these same individuals will have a subsequent blood lead level analysis. This observational study will provide the data necessary to determine the washout period and sample size for a prospective interventional study to evaluate if meat harvested with lead bullets raises blood-lead levels in those who consume the meat. This study has been approved by the Health and Disabilities Ethics Committees of New Zealand. TRIAL REGISTRATION: NCT02775890.

Hepatocytes detoxify Atuna racemosa extract.

A number of traditional medicine plants are hepatotoxic. Thus, while the traditional uses of Atuna racemosa suggest little indication for toxicity, it is nonetheless important to examine the potential for this extract to target the liver. Using Jurkat T cells and HepG2 hepatocytes as a model, the potential hepatotoxicity of this extract was evaluated. The results of a conditioned media experiment suggest that A. racemosa extract would likely be detoxified by the liver. These results provide the necessary background to initiate an in vivo toxicology investigation.

Little risk of severe complications associated with Zika infection in New Zealand.

Zika virus infection has raised considerable concern in New Zealand, but the risks faced by most New Zealanders, while real, are quite small as New Zealand does not harbor the primary mosquito vector. Furthermore, in individuals with a competent immune system, the acute illness caused by Zika virus infection is generally mild. Serious complication associated with Zika virus infections include microcephaly and Guillain-Barré Syndrome. Pacific Island countries have reported cases of Zika virus infection and these climates support the mosquito vector. Thus, travelers to these areas are at risk of infection. New Zealand travelers returning from endemic areas have developed the illness associated with the virus, but the probability of autochthonous transmission in New Zealand is very small.