Ethical considerations in the regulation and use of herbal medicines in the European Union.

The regulation and use of herbal medicines is a topic of debate due to concerns about their quality, safety, and efficacy. EU Directive 2004/24/EC on Herbal Medicinal Products was a significant step towards establishing a regulatory framework for herbal medicinal products in the EU, and bridging the gap between conventional and herbal medicines. This Directive allows herbal medicinal products to be marketed in the EU through full marketing authorisation, well-established use, and traditional use of herbal medicinal products. The framework relies on the correlation between the therapeutic claims of herbal medicine and the scientific evidence backing them up: the greater the claims made regarding medicinal benefits, the more evidence is required to substantiate its efficacy and safety. This regulatory framework acknowledges and incorporates traditional knowledge when evaluating herbal medicines, showcasing a balanced approach that values cultural traditions while mandating monographs for traditional herbal medicinal products. Excluding herbal medicines completely limits access to affordable treatment, particularly when they serve as the only alternative for some, and protects consumer autonomy. This EU framework could therefore serve as a practical guidance for the use and regulation of herbal medicines, even outside the EU. In conclusion, it is argued that the same moral imagination and courage shown by regulators in the case of herbal medicines could perhaps be used in the regulatory frameworks of other healthcare products.

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses.

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8(+) T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8(+) T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8(+) T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8(+) T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.

Investigating the role for IL-21 in rabies virus vaccine-induced immunity.

Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R-/-) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R-/- mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R-/- mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R-/- mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.

Protective vaccine-induced CD4(+) T cell-independent B cell responses against rabies infection.

A major goal in rabies virus (RV) research is to develop a single-dose postexposure prophylaxis (PEP) that would simplify vaccination protocols, reduce costs associated with rabies prevention in humans, and save lives. Live replication-deficient RV-based vaccines are emerging as promising single-dose vaccines to replace currently licensed inactivated RV-based vaccines. Nonetheless, little is known about how effective B cells develop in response to live RV-based vaccination. Understanding this fundamental property of rabies immunology may help in developing a single-dose RV vaccine. Typically, vaccines induce B cells secreting high-affinity, class-switched antibodies during germinal center (GC) reactions; however, there is a lag time between vaccination and the generation of GC B cells. In this report, we show that RV-specific antibodies are detected in mice immunized with live but not inactivated RV-based vaccines before B cells displaying a GC B cell phenotype (B220(+)GL7(hi)CD95(hi)) are formed, indicating a potential role for T cell-independent and early extrafollicular T cell-dependent antibody responses in the protection against RV infection. Using two mouse models of CD4(+) T cell deficiency, we show that B cells secreting virus-neutralizing antibodies (VNAs) are induced via T cell-independent mechanisms within 4 days postimmunization with a replication-deficient RV-based vaccine. Importantly, mice that are completely devoid of T cells (B6.129P2-Tcrß(tm1Mom) Tcrδ(tm1Mom)/J) show protection against pathogenic challenge shortly after immunization with a live replication-deficient RV-based vaccine. We show that vaccines that can exploit early pathways of B cell activation and development may hold the key for the development of a single-dose RV vaccine wherein the rapid induction of VNA is critical.

Tau Ser262 phosphorylation is critical for Abeta42-induced tau toxicity in a transgenic Drosophila model of Alzheimer's disease.

The amyloid-beta 42 (Abeta42) peptide has been suggested to promote tau phosphorylation and toxicity in Alzheimer's disease (AD) pathogenesis; however, the underlying mechanisms are not fully understood. Using transgenic Drosophila expressing both human Abeta42 and tau, we show here that tau phosphorylation at Ser262 plays a critical role in Abeta42-induced tau toxicity. Co-expression of Abeta42 increased tau phosphorylation at AD-related sites including Ser262, and enhanced tau-induced neurodegeneration. In contrast, formation of either sarkosyl-insoluble tau or paired helical filaments was not induced by Abeta42. Co-expression of Abeta42 and tau carrying the non-phosphorylatable Ser262Ala mutation did not cause neurodegeneration, suggesting that the Ser262 phosphorylation site is required for the pathogenic interaction between Abeta42 and tau. We have recently reported that the DNA damage-activated Checkpoint kinase 2 (Chk2) phosphorylates tau at Ser262 and enhances tau toxicity in a transgenic Drosophila model. We detected that expression of Chk2, as well as a number of genes involved in DNA repair pathways, was increased in the Abeta42 fly brains. The induction of a DNA repair response is protective against Abeta42 toxicity, since blocking the function of the tumor suppressor p53, a key transcription factor for the induction of DNA repair genes, in neurons exacerbated Abeta42-induced neuronal dysfunction. Our results demonstrate that tau phosphorylation at Ser262 is crucial for Abeta42-induced tau toxicity in vivo, and suggest a new model of AD progression in which activation of DNA repair pathways is protective against Abeta42 toxicity but may trigger tau phosphorylation and toxicity in AD pathogenesis.

A DNA damage-activated checkpoint kinase phosphorylates tau and enhances tau-induced neurodegeneration.

Hyperphosphorylation of the microtubule associated protein tau is detected in the brains of individuals with a range of neurodegenerative diseases including Alzheimer's disease (AD). An imbalance in phosphorylation and/or dephosphorylation of tau at disease-related sites has been suggested to initiate the abnormal metabolism and toxicity of tau in disease pathogenesis. However, the mechanisms underlying abnormal phosphorylation of tau in AD are not fully understood. Here, we show that the DNA damage-activated Checkpoint kinase 2 (Chk2) is a novel tau kinase and enhances tau toxicity in a transgenic Drosophila model. Overexpression of Drosophila Chk2 increases tau phosphorylation at Ser262 and enhances tau-induced neurodegeneration in transgenic flies expressing human tau. The non-phosphorylatable Ser262Ala mutation abolishes Chk2-induced enhancement of tau toxicity, suggesting that the Ser262 phosphorylation site is involved in the enhancement of tau toxicity by Chk2. In vitro kinase assays revealed that human Chk2 and a closely related checkpoint kinase 1 (Chk1) directly phosphorylate human tau at Ser262. We also demonstrate that Drosophila Chk2 does not modulate the activity of the fly homolog of microtubule affinity regulating kinase, which has been shown to be a physiological tau Ser262 kinase. Since accumulation of DNA damage has been detected in the brains of AD patients, our results suggest that the DNA damage-activated kinases Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in the pathogenesis of AD.

Mitochondrial mislocalization underlies Abeta42-induced neuronal dysfunction in a Drosophila model of Alzheimer's disease.

The amyloid-beta 42 (Abeta42) is thought to play a central role in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms by which Abeta42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Abeta42 induces mitochondrial mislocalization, which contributes to Abeta42-induced neuronal dysfunction in a transgenic Drosophila model. In the Abeta42 fly brain, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. In contrast, organization of microtubule or global axonal transport was not significantly altered at this stage. Abeta42-induced behavioral defects were exacerbated by genetic reductions in mitochondrial transport, and were modulated by cAMP levels and PKA activity. Levels of putative PKA substrate phosphoproteins were reduced in the Abeta42 fly brains. Importantly, perturbations in mitochondrial transport in neurons were sufficient to disrupt PKA signaling and induce late-onset behavioral deficits, suggesting a mechanism whereby mitochondrial mislocalization contributes to Abeta42-induced neuronal dysfunction. These results demonstrate that mislocalization of mitochondria underlies the pathogenic effects of Abeta42 in vivo.

Overexpression of neprilysin reduces alzheimer amyloid-beta42 (Abeta42)-induced neuron loss and intraneuronal Abeta42 deposits but causes a reduction in cAMP-responsive element-binding protein-mediated transcription, age-dependent axon pathology, and premature death in Drosophila.

The amyloid-beta42 (Abeta42) peptide has been suggested to play a causative role in Alzheimer disease (AD). Neprilysin (NEP) is one of the rate-limiting Abeta-degrading enzymes, and its enhancement ameliorates extracellular amyloid pathology, synaptic dysfunction, and memory defects in mouse models of Abeta amyloidosis. In addition to the extracellular Abeta, intraneuronal Abeta42 may contribute to AD pathogenesis. However, the protective effects of neuronal NEP expression on intraneuronal Abeta42 accumulation and neurodegeneration remain elusive. In contrast, sustained NEP activation may be detrimental because NEP can degrade many physiological peptides, but its consequences in the brain are not fully understood. Using transgenic Drosophila expressing human NEP and Abeta42, we demonstrated that NEP efficiently suppressed the formation of intraneuronal Abeta42 deposits and Abeta42-induced neuron loss. However, neuronal NEP overexpression reduced cAMP-responsive element-binding protein-mediated transcription, caused age-dependent axon degeneration, and shortened the life span of the flies. Interestingly, the mRNA levels of endogenous fly NEP genes and phosphoramidon-sensitive NEP activity declined during aging in fly brains, as observed in mammals. Taken together, these data suggest both the protective and detrimental effects of chronically high NEP activity in the brain. Down-regulation of NEP activity in aging brains may be an evolutionarily conserved phenomenon, which could predispose humans to developing late-onset AD.

Abeta42 mutants with different aggregation profiles induce distinct pathologies in Drosophila.

Aggregation of the amyloid-beta-42 (Abeta42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer's disease (AD), and the prevention of Abeta aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Abeta can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Abeta42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Abeta42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Abeta42Arc) and an artificial mutation (Abeta42art) that is known to suppress aggregation and toxicity of Abeta42 in vitro. In the Drosophila brain, Abeta42Arc formed more oligomers and deposits than did wild type Abeta42, while Abeta42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Abeta peptides. Surprisingly, however, Abeta42art caused earlier onset of memory defects than Abeta42. More remarkably, each Abeta induced qualitatively different pathologies. Abeta42Arc caused greater neuron loss than did Abeta42, while Abeta42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Abeta aggregates: Abeta42Arc formed large deposits in the cell body, Abeta42art accumulated preferentially in the neurites, while Abeta42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Abeta42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.