Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking.

Biodiversity is defined as the presence of a variety of living organisms on the Earth that is essential for human survival. However, anthropogenic activities are causing the sixth mass extinction, threatening even our own species. For many animals, dwindling numbers are becoming fragmented populations with low genetic diversity, threatening long-term species viability. With extinction rates 1000-10,000 times greater than natural, ex situ and in situ conservation programmes need additional support to save species. The indefinite storage of cryopreserved (-196°C) viable cells and tissues (cryobanking), followed by assisted or advanced assisted reproductive technology (ART: utilisation of oocytes and spermatozoa to generate offspring; aART: utilisation of somatic cell genetic material to generate offspring), may be the only hope for species' long-term survival. As such, cryobanking should be considered a necessity for all future conservation strategies. Following cryopreservation, ART/aART can be used to reinstate lost genetics back into a population, resurrecting biodiversity. However, for this to be successful, species-specific protocol optimisation and increased knowledge of basic biology for many taxa are required. Current ART/aART is primarily focused on mammalian taxa; however, this needs to be extended to all, including to some of the most endangered species: amphibians. Gamete, reproductive tissue and somatic cell cryobanking can fill the gap between losing genetic diversity today and future technological developments. This review explores species prioritisation for cryobanking and the successes and challenges of cryopreservation and multiple ARTs/aARTs. We here discuss the value of cryobanking before more species are lost and the potential of advanced reproductive technologies not only to halt but also to reverse biodiversity loss. Lay summary: The world is undergoing its sixth mass extinction; however, unlike previous events, the latest is caused by human activities and is resulting in the largest loss of biodiversity (all living things on Earth) for 65 million years. With an extinction rate 1000-10,000-fold greater than natural, this catastrophic decline in biodiversity is threatening our own survival. As the number of individuals within a species declines, genetic diversity reduces, threatening their long-term existence. In this review, the authors summarise approaches to indefinitely preserve living cells and tissues at low temperatures (cryobanking) and the technologies required to resurrect biodiversity. In the future when appropriate techniques become available, these living samples can be thawed and used to reinstate genetic diversity and produce live young ones of endangered species, enabling their long-term survival. The successes and challenges of genome resource cryopreservation are discussed to enable a move towards a future of stable biodiversity.

A novel phospholipid-based drug formulation, VP025, modulates age- and LPS-induced microglial activity in the rat.

BACKGROUND: A common change that occurs with age in the central nervous system is an increase in microglial-associated inflammation. This is usually coupled with an increase in the concentration of the inflammatory cytokine interleukin-1beta (IL-1beta) in the hippocampus and an inhibition in long-term potentiation. OBJECTIVES: To assess the effects of a novel preparation of phospholipid nanoparticles incorporating phosphatidylglycerol, VP025, on inflammatory changes in hippocampus of aged and lipopolysaccharide (LPS)-treated rats. METHODS/RESULTS: We report that a possible initial target cell of the putative anti-inflammatory actions of VP025 may be macrophages, as VP025 is engulfed by, and has the capacity to alter the activity of, these cells. VP025 reversed the increase in IFN-gamma concentration in supernatant taken from peritoneal macrophages harvested from LPS-treated rats. In addition, markers of microglial activity, major histocompatibility complex class II (MHC II) mRNA expression, CD40 expression and IL-1beta concentration were increased, and CD200 expression was reduced, in the hippocampus of these rats. VP025 reversed changes in CD40, IL-1beta and CD200 in aged rats, and also restored long-term potentiation in aged and LPS-treated rats. CONCLUSIONS: We conclude that VP025 has the ability to modulate the activity of macrophage, microglia and neurons in response to stressors such as ageing and LPS treatment.

The deficit in long-term potentiation induced by chronic administration of amyloid-beta is attenuated by treatment of rats with a novel phospholipid-based drug formulation, VP025.

Amyloid-beta (Abeta) peptides, the primary component of the amyloid plaques in Alzheimer's disease (AD), exert profound effects on neurons in vitro and negatively impact on neuronal function in vivo. One of the consequences of increased Abeta in the brain, either as a result of overexpression of the precursor amyloid precursor protein in transgenic mice, or injection into the brain is a decrease in one form of synaptic plasticity, long-term potentiation (LTP) in the hippocampus. Here we investigated the effect of infusion of Abeta for 28 days on LTP in dentate gyrus of rats and demonstrate that it was profoundly decreased compared with control-treated rats. We show that this effect is accompanied by increased activity of caspase 3, which is an indicator of cell stress. Significantly these changes were attenuated in animals which were pretreated with particles incorporating phosphatidylglycerol (VP025) and the evidence indicated that even when treatment was given 2 weeks after the start of the Abeta infusion, VP025 was capable of attenuating Abeta-induced changes. The evidence suggests that activation of caspase 3 was mediated by an Abeta-induced increase in sphingomyelinase, with the subsequent production of ceramide which is known to have a detrimental effect on neuronal function.

Treatment with phosphotidylglycerol-based nanoparticles prevents motor deficits induced by proteasome inhibition: implications for Parkinson's disease.

Failure of the ubiquitin-proteasome system to degrade abnormal proteins may underlie the accumulation of alpha-synuclein and dopaminergic neuronal degeneration that occurs in Parkinson's disease. Consequently, a reduction of functional proteasome activity has been implicated in Parkinson's disease. VP025 (Vasogen Inc.) is a preparation of phospholipid nanoparticles incorporating phosphatidylglycerol that has been shown to have neuroprotective effects. We show that VP025 prevents the deficits in motor coordination and dopamine observed in a proteasome inhibitor rat model of PD. Thus, VP025 may have a therapeutic effect on the impairment of dopaminergic-mediated motor activity induced by proteasome inhibition.

Biologic effects and basic science of a novel immune-modulation therapy.

The immune system is a system of dynamic equilibrium, with inflammatory responses (mediated by T helper type 1 cells, interleukin [IL]-1beta, interferon-gamma, and tumor necrosis factor-alpha [TNF-alpha]) being balanced by anti-inflammatory responses (mediated by T regulatory type 1 cell, T helper type 3 cells, IL-4, IL-10, and transforming growth factor-beta). Therefore, neutralization of inappropriate inflammatory cytokines is a therapeutic strategy that has been attempted in many chronic inflammatory conditions, mostly targeting TNF-alpha, using either monoclonal antibodies or modified receptor proteins (etanercept). There is functional redundancy among the inflammatory cytokines. For example, in addition to TNF-alpha, both IL-1beta and IL-6 are elevated in patients with chronic heart failure (CHF); thus neutralizing the activity of TNF-alpha alone may be an inadequate approach in this patient group. Immune-modulation therapy (IMT) results in downregulation of proinflammatory cytokine levels and upregulation of anti-inflammatory cytokines. This alteration in the balance between proinflammatory and anti-inflammatory cytokines may be more appropriate than neutralizing the activity of a single cytokine in the treatment of conditions such as CHF. Several animal studies investigating the effect of IMT in inflammatory conditions including allergic contact hypersensitivity, ischemia reperfusion injury, and atherogenesis are reviewed.

Evidence of an anti-inflammatory role for Vasogen's immune modulation therapy.

We have reported that Vasogen's immune modulation therapy (IMT), a procedure involving intramuscular administration of autologous/syngeneic blood, which has been exposed ex vivo to increased temperature, UVC light and oxidation, prevents several LPS-induced inflammatory changes in the hippocampus. Here, we investigated neuroprotective effects of IMT in cortical tissue, and report that the treatment acts as an anti-inflammatory and antioxidative agent, reducing the concentration of TNFalpha and the accumulation of reactive oxygen species. The data couple these changes with an increase in the concentration of the anti-inflammatory cytokine IL-10, and a decrease in activation of the stress-activated protein kinase, c-jun N-terminal kinase. Consistent with these putative protective effects of IMT, we report that the LPS-induced increase in TUNEL staining, which is indicative of cell death, is prevented by IMT.

Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus.

It is well documented that long term potentiation (LTP) is impaired in the hippocampus of the aged animal. Among the changes that contribute to this impairment is an increase in hippocampal concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and increased IL-1beta-induced signaling. In this study we investigated the possibility that these changes were a consequence of decreased concentration of the anti-inflammatory cytokine, IL-4, and decreased IL-4-stimulated signaling. We report that functional IL-4 receptors are expressed on granule cells of the dentate gyrus and that receptor activation results in phosphorylation of JAK1 and STAT6. Hippocampal IL-4 concentration was decreased with age, and this was accompanied by a decrease in phosphorylation of JAK1 and STAT6. The evidence indicates that IL-4 modulates expression of IL-1beta mRNA and protein and that it attenuates IL-1beta-induced impairment of LTP and phosphorylation of JNK and c-Jun. We argued that, if a decrease in hippocampal IL-4 concentration significantly contributed to the age-related impairment in LTP, then restoration of IL-4 should restore LTP. To test this, we treated rats with VP015 (phospholipid microparticles-incorporating phosphatidylserine), which increases IL-4 concentration in hippocampus. The data indicate that the VP015-induced increase in IL-4 concentration in hippocampus of aged rats and lipopolysaccharide (LPS)-treated rats was accompanied by a reversal of the age-related and LPS-induced impairment in LTP in perforant path granule cell synapses. We propose that interplay between pro-inflammatory and anti-inflammatory responses impact significantly on synaptic function in the hippocampus of the aged rat.

Renal ischemia-reperfusion injury in the rat is prevented by a novel immune modulation therapy.

BACKGROUND: Vasogen Inc.'s (Mississauga, Ontario, Canada) immune modulation therapy (IMT) is a therapy in which cells from the patient's own blood are modified by ex vivo exposure to specific physicochemical stressors, including oxidation, ultraviolet (UV) light, and an elevated temperature. The therapy has been shown to have a beneficial effect in models of inflammation and vascular diseases. This study tested the hypothesis that IMT can prevent renal ischemia-reperfusion (I/R) injury in rats. METHODS: Whole blood was collected from syngeneic age-matched donors by cardiac puncture. It was treated with a combination of controlled physiochemical stressors consisting of elevated temperature, a gas mixture of medical oxygen containing ozone, and UV light. The treated blood (150 microL) was injected in the gluteal muscle. Control animals received the same volume of untreated blood or physiological saline. Transient (45 or 60 minutes) left-renal ischemia was produced with simultaneous contralateral nephrectomy in treated and control spontaneously hypertensive rats (SHR). Young and old male and female rats were studied. Plasma creatinine, diuresis, and the survival rates of each group were compared. Renal apoptosis-necrosis was estimated by DNA laddering, histology, and in situ terminal deoxynucleotidyl transferase assay. mRNA levels of several regulators of apoptosis-regeneration were determined in control and postischemic kidneys by Northern blotting. RESULTS: IMT pretreatment of SHR significantly reduced renal I/R injury compared with equivalent placebo treatments consisting of untreated blood- or saline-injected SHR, as evidenced by a significant increase of the survival rate curves in young and old male SHR, which correlated with 24-hour postischemic diuresis. The increases in plasma creatinine following renal I/R were significantly lower in IMT-treated young male and old female SHR compared with saline or untreated blood-injected controls. Dilution analysis showed that the protective effect of treated blood was lost by dilution. Loss of epithelial cells was reduced in IMT-treated rats, with a significant decline in the peak of apoptosis 12 hours after acute ischemic renal injury. IMT did not modify the pattern of mRNA levels of several genes involved in the inflammation and regeneration processes. CONCLUSION: Our data demonstrate that IMT prevents the destruction of kidney tissue and the resulting animal death caused by renal I/R injury.

Attenuation of LPS-induced changes in synaptic activity in rat hippocampus by Vasogen's Immune Modulation Therapy.

Systemic injection of lipopolysaccharide (LPS) blocks the expression of long-term potentiation in the hippocampus of the rat. This is coupled with increased IL-1beta concentration and c-Jun NH(2)-terminal kinase activity, as well as an increase in the number of cells displaying apoptotic characteristics in the hippocampus. Vasogen's Immune Modulation Therapy (IMT) is a procedure involving intramuscular administration of syngeneic blood which has been exposed ex vivo to elevated temperature, oxidation and ultraviolet light. We report that Vasogen's IMT significantly abrogates these LPS-induced effects with a concomitant increase in the concentration of the anti-inflammatory cytokine IL-10. These data suggest that Vasogen's IMT may play a protective role against the deleterious effects of immune insults in the brain.