Weight of evidence evaluation for chemical-induced immunotoxicity for PFOA and PFOS: findings from an independent panel of experts.

Immunotoxicity is the critical endpoint used by some regulatory agencies to establish toxicity values for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). However, the hypothesis that exposure to certain per- and polyfluoroalkyl substances (PFAS) causes immune dysregulation is subject to much debate. An independent, international expert panel was engaged utilizing methods to reduce bias and "groupthink". The panel concluded there is moderate evidence that PFOS and PFOA are immunotoxic, based primarily on evidence from animal data. However, species concordance and human relevance cannot be well established due to data limitations. The panel recommended additional testing that includes longer-term exposures, evaluates both genders, includes other species of animals, tests lower dose levels, assesses more complete measures of immune responses, and elucidates the mechanism of action. Panel members agreed that the Faroe Islands cohort data should not be used as the primary basis for deriving PFAS risk assessment values. The panel agreed that vaccine antibody titer is not useful as a stand-alone metric for risk assessment. Instead, PFOA and PFOS toxicity values should rely on multiple high-quality studies, which are currently not available for immune suppression. The panel concluded that the available PFAS immune epidemiology studies suffer from weaknesses in study design that preclude their use, whereas available animal toxicity studies provide comprehensive dataset to derive points of departure (PODs) for non-immune endpoints. The panel recommends accounting for potential PFAS immunotoxicity by applying a database uncertainty factor to POD values derived from animal studies for other more robustly supported critical effects.

Parasite load stemming from immunization route determines the duration of liver-stage immunity.

Immunization with radiation-attenuated Plasmodium sporozoites (RAS) induces sterile and long-lasting protective immunity. Although intravenous (IV) route of RAS immunization is reported to induce superior immunity compared to intradermal (ID) injection, its role in the maintenance of sterile immunity is yet to be understood. We investigated whether the route of homologous sporozoite challenge of Plasmodium berghei (Pb) RAS-immunized mice would influence the longevity of protection. C57BL/6 mice immunized with Pb-RAS by IV were 100% protected upon primary IV/ID sporozoite challenge. In contrast, ID immunization resulted in 80% protection, regardless of primary challenge route. Interestingly, the route of primary challenge was found to bring difference in the maintenance of sterile protection. While IV Pb RAS-immunized mice remained protected at all challenges regardless of the route of primary challenge, ID Pb-RAS-immunized mice receiving ID primary challenge became parasitaemic upon secondary IV challenge. Significantly, primary IV challenge of Pb RAS ID-immunized mice resulted in 80% and 50% survival at secondary and tertiary challenges, respectively. According to phenotypically diverse liver CD8+ T cells, the percentages and the numbers of both CD8+ T effector memory and resident memory cells were significantly higher in IV than in ID Pb RAS-immunized mice. IFN-γ-producing CD8+ T cells specific to Pb TRAP130 and MIP-4-Kb-17 were also found significantly higher in IV mice than in ID mice. The enhanced T-cell generation and the longevity of protection appear to be dependent on the parasite load during challenge when infection is tolerated under suboptimal CD8+ T-cell response.

High-level artemisinin-resistance with quinine co-resistance emerges in P. falciparum malaria under in vivo artesunate pressure.

BACKGROUND: Humanity has become largely dependent on artemisinin derivatives for both the treatment and control of malaria, with few alternatives available. A Plasmodium falciparum phenotype with delayed parasite clearance during artemisinin-based combination therapy has established in Southeast Asia, and is emerging elsewhere. Therefore, we must know how fast, and by how much, artemisinin-resistance can strengthen. METHODS: P. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2Rγ-/-immunocompromised mice, with progressive dose increments as parasites recovered. The parasite's response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro. RESULTS: Artemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of > 3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC50 remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2-11.2) vs. ART-R30: 11.5 nM (6.6-16.9), F = 0.525, p = 0.47). However, when in vivo resistance strengthened further, IC50 increased 10-fold (ART-R240 100.3 nM (92.9-118.4), F = 304.8, p < 0.0001), reaching a level much higher than ever seen in clinical samples. Artemisinin resistance in this African P. falciparum strain was not associated with mutations in kelch-13, casting doubt over the universality of this genetic marker for resistance screening. Remarkably, despite exclusive exposure to artesunate, full resistance to quinine, the only other drug sufficiently fast-acting to deal with severe malaria, evolved independently in two parasite lines exposed to different artesunate regimens in vivo, and was confirmed in vitro. CONCLUSION: P. falciparum has the potential to evolve extreme artemisinin resistance and more complex patterns of multidrug resistance than anticipated. If resistance in the field continues to advance along this trajectory, we will be left with a limited choice of suboptimal treatments for acute malaria, and no satisfactory option for severe malaria.

Chitosan Nanoparticles of Gamma-Oryzanol: Formulation, Optimization, and In vivo Evaluation of Anti-hyperlipidemic Activity.

The elevated blood levels of cholesterol and low-density lipoproteins result in hyperlipidemia. The available expensive prophylactic treatments are kindred with severe side effects. Therefore, we fabricated the polymeric nanoparticles of gamma-oryzanol to achieving the improved efficacy of drug. The nanoparticles were prepared by ionic gelation method and optimized using 23 full factorial design taking drug/polymer ratio (X1), polymer/cross linking agent ratio (X2), and stirring speed (X3) as independent variables. The average particle size, percentage entrapment efficiency, and in vitro drug release at 2, 12, and 24 h were selected as response parameters. The factorial batches were statistically analyzed and optimized. The optimized nanoparticles were characterized with respect to particle size (141 nm) and zeta potential (+ 6.45 mV). Results obtained with the prepared and characterized formulation showed 83% mucoadhesion towards the intestinal mucosa. The in vitro findings were complemented well by in vivo anti-hyperlipidemic activity of developed formulation carried out in Swiss albino mouse model. The in vivo studies showed improved atherogenic index, malondialdehyde, and superoxide dismutase levels in poloxamer-407-induced hyperlipidemic animals when treated with oryzanol and gamma-oryzanol nanoformulation. Based on our findings, we believe that chitosan-mediated delivery of gamma-oryzanol nanoparticles might prove better in terms of anti-hyperlipidemic therapeutics.

Functionalized Lipid-Polymer Hybrid Nanoparticles Mediated Codelivery of Methotrexate and Aceclofenac: A Synergistic Effect in Breast Cancer with Improved Pharmacokinetics Attributes.

The present study was aimed to coencapsulate methotrexate (MTX) and aceclofenac (ACL) in fucose anchored lipid-polymer hybrid nanoparticles (Fu-LPHNPs) to achieve target specific and controlled delivery for developing therapeutic interventions against breast cancer. The effective combination therapy requires coadministration of drugs to achieve synergistic effect on tumor with minimum adverse effects. Present study investigates the potential of codelivery of MTX and ACL through LPHNPs in MCF-7 and triple negative breast cancer cells (MDA-MB-231). We obtained LPHNPs in the nanosize range (<150 nm) with better particle size distribution (<0.3). The entrapment and loading efficiency of MTX and ACL was calculated as 85-90% and 10-12%, respectively. The coumarin-6 LPHNP formulations showed rapid internalization within 2 h incubation with MCF-7 and MDA-MB-231 cells. With 8-10 times, greater bioavailability of drug-loaded LPHNPs than free MTX and ACL was obtained. Also, antitumor efficacy of MTX- and ACL-loaded LPHNPs was determined on DMBA-induced experimental breast cancer mouse model. This model showed better control over tumor growth with MTX- and ACL-loaded LPHNPs than the combination of MTX and ACL or MTX alone. ACL-loaded LPHNPs showed prophylactic and anticancer activity in DMBA-induced mouse model at higher dose (10 mg/kg). ACL-LPHNPs confer synergistic anticancer effect when administered in combination with MTX. In conclusion, ACL enhances the therapeutic and anticancer efficacy of MTX, when coencapsulated into fucose-anchored LPHNPs, as confirmed by cell viability and serum angiogenesis (IL-6, TNF-α, IL-1ß, COX2, and MMP1) at both transcript and proteome level.

The ligand (s) anchored lipobrid nanoconstruct mediated delivery of methotrexate: an effective approach in breast cancer therapeutics.

The present study was designed to engineer surface-anchored and methotrexate loaded lipobrid nano-constructs for targeting breast cancer. Ligands (fucose, galactose and mannose) anchored lipobrid nano-constructs were used to compare and assess delivery efficiency in breast cancer cell lines as well as in DMBA induced breast cancer animal model. The developed and characterized formulations were used to comparatively assess cellular uptake, cell-viability, apoptosis, lysosomal membrane permeability, bioavailability, bio-distribution, changes in tumor volume and animal survival. Our results show greater cellular uptake, cytotoxicity at low IC50, apoptosis with altered lysosomal membrane permeability and greater rate of degradation of lysosomal membrane. We saw better bioavailability and tumor targeting efficiency with minimum secondary organ drug distribution. The significant reduction was seen in tumor burden with ligand anchored lipobrids in comparison to plain and MTX-lipobrid formulations. In conclusion, fucose anchored MTX-lipobrid formulation showed promising results, and warrants to explore the development of therapeutic interventions for breast cancer.

A synergistic approach of adapalene-loaded nanostructured lipid carriers, and vitamin C co-administration for treating acne.

The present study documents the fabrication and characterization of a topically applicable gel loaded with nanostructured lipid carriers (NLCs) of adapalene (ADA) and vitamin C (ascorbyl-6-palmitate [AP]). The NLCs were prepared by high pressure homogenization (HPH) method followed by incorporation into AP loaded gel. The fabricated system was characterized for size, poly dispersity index, entrapment efficiency (EE) and in vitro drug release properties, and was further investigated for skin compliance, skin transport characteristics (skin permeation and bio-distribution), rheological behavior, texture profile analysis and anti-acne therapeutic potential against testosterone-induced acne in male Wistar rats. The NLC-based formulation improved targeting of the skin epidermal layer and reducing systemic penetration. The co-administration of vitamin C led to an adjunct effect in acne therapy in physiological conditions. In brief, the present results suggest the potential of NLCs as a novel carrier for the dermal delivery of ADA and also the synergistic effect of vitamin C in topical therapeutics.

Protocol for oleuropein-induced autophagy mediating drug tolerance in P. falciparum.

The anti-inflammatory activity of a phytocompound (oleuropein [OLP]) in the lipopolysaccharide (LPS)-mimicked macrophage model of inflammation demonstrates the importance of PI3K-Akt1 signaling in establishing "immune homeostasis." Here, we present a protocol for the cultivation of in vitro cultures of P. falciparum for carrying out drug sensitivity assays. We describe steps for parasite synchronization, drug treatment, DNA isolation, and starvation-induced autophagy. This protocol provides insights into autophagy and parasite tolerance to drug pressure. For complete details on the use and execution of this protocol, please refer to Sharma et al.1.

Oleuropein mediated autophagy begets antimalarial drug resistance.

Drug resistance in Plasmodium falciparum presents a formidable challenge to the humanity. And, unavailability of an effective vaccine worsens the situation further. Autophagy is one of the mechanisms employed by parasite to evade drug pressure to survive. Autophagy induced by the P. falciparum in response to the oleuropein pressure may answer many questions related to the parasite survival as well as evolving drug tolerance. The survival/autophagy axis could be an important avenue to explore in order to address certain questions related to the evolution of drug resistance. In addition, humanized mouse model of P. falciparum infection could serve as an important preclinical tool to investigate the oleuropein-induced autophagy, potentially helping to dissect the mechanisms underlying the development of antimalarial drug resistance.

Poly(I:C) and R848 ligands show better adjuvanticity to induce B and T cell responses against the antigen(s).

Poly(I:C) and R848, synthetic ligands that activate Toll-like receptor 3 (TLR3) and TLR7/8 respectively, have been well-established for their ability to stimulate the immune system and induce antigen-specific immune responses. These ligands are capable of inducing the production of cytokines and chemokines, and hence support the activation and differentiation of B and T cells. We saw the long-lasting and perdurable immune responses by these adjuvants essentially required for an efficacious subunit vaccine. In this study, we investigated the potential of poly(I:C) and R848 to elicit B and T cell responses to the OVA antigen. We assessed the stimulatory effects of these ligands on the immune system, their impact on B and T cell activation, and their ability to enhanced generation of B and T cells. Collectively, our findings contribute to the understanding how poly(I:C) and R848 can be utilized as an adjuvant system to enhance immune responses to protein-based subunit vaccines. In the end, this work provides insights for the development of novel vaccination strategies and improving the vaccine efficacy. Present work shall help formulate newer strategies for subunit vaccines to address the infectious diseases.

Biogenically synthesized green silver nanoparticles exhibit antimalarial activity.

The suboptimal efficacies of existing anti-malarial drugs attributed to the emergence of drug resistance dampen the clinical outcomes. Hence, there is a need for developing novel drug and drug targets. Recently silver nanoparticles (AgNPs) constructed with the leaf extracts of Euphorbia cotinifolia were shown to possess antimalarial activity. Therefore, the synthesized AgNPs from Euphorbia cotinifolia (EcAgNPs) were tested for their parasite clearance activity. We determined the antimalarial activity in the asexual blood stage infection of 3D7 (laboratory strain) P. falciparum. EcAgNPs demonstrated the significant inhibition of parasite growth (EC50 of 0.75 µg/ml) in the routine in vitro culture of P. falciparum. The synthesized silver nanoparticles were seen to induce apoptosis in P. falciparum through increased reactive oxygen species (ROS) ROS production and activated programmed cell death pathways characterized by the caspase-3 and calpain activity. Also, altered transcriptional regulation of Bax/Bcl-2 ratio indicated the enhanced apoptosis. Moreover, inhibited expression of PfLPL-1 by EcAgNPs is suggestive of the dysregulated host fatty acid flux via parasite lipid storage. Overall, our findings suggest that EcAgNPs are a non-toxic and targeted antimalarial treatment, and could be a promising therapeutic approach for clearing malaria infection.

Co-Delivery of Aceclofenac and Methotrexate Nanoparticles Presents an Effective Treatment for Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) is a common acute inflammatory autoimmune connective tissue arthropathy. The genetic studies, tissue analyses, experimental animal models, and clinical investigations have confirmed that stromal tissue damage and pathology driven by RA mounts the chronic inflammation and dysregulated immune events. Methods: We developed methotrexate (MTX)-loaded lipid-polymer hybrid nanoparticles (MTX-LPHNPs) and aceclofenac (ACE)-loaded nanostructured lipid carriers (ACE-NLCs) for the efficient co-delivery of MTX and ACE via intravenous and transdermal routes, respectively. Bio-assays were performed using ex-vivo skin permeation and transport, macrophage model of inflammation (MMI) (LPS-stimulated THP-1 macrophages), Wistar rats with experimental RA (induction of arthritis with Complete Freund's adjuvant; CFA and BCG), and programmed death of RA affected cells. In addition, gene transcription profiling and serum estimation of inflammatory, signaling, and cell death markers were performed on the blood samples collected from patients with RA. Results: Higher permeation of ACE-NLCs/CE across skin layers confirming the greater "therapeutic index" of ACE. The systemic delivery of MTX-loaded LPHNPs via the parenteral (intravenous) route is shown to modulate the RA-induced inflammation and other immune events. The regulated immunological and signaling pathway(s) influence the immunological axis to program the death of inflamed cells in the MMI and the animals with the experimental RA. Our data suggested the CD40-mediated and Akt1 controlled cell death along with the inhibited autophagy in vitro. Moreover, the ex vivo gene transcription profiling in drug-treated PBMCs and serum analysis of immune/signalling markers confirmed the therapeutic role co-delivery of drug nanoparticles to treat RA. The animals with experimental RA receiving drug treatment were shown to regain the structure of paw bones and joints similar to the control and were comparable with the market formulations. Conclusion: Our findings confirmed the use of co-delivery of drug nanoformulations as the "combination drug regimen" to treat RA.

Oleuropein activates autophagy to circumvent anti-plasmodial defense.

Antimalarial drug resistance and unavailability of effective vaccine warrant for newer drugs and drug targets. Hence, anti-inflammatory activity of phyto-compound (oleuropein; OLP) was determined in antigen (LPS)-stimulated human THP-1 macrophages (macrophage model of inflammation; MMI). Reduction in the inflammation was controlled by the PI3K-Akt1 signaling to establish the "immune-homeostasis." Also, OLP treatment influenced the cell death/autophagy axis leading to the modulated inflammation for extended cell survival. The findings with MII prompted us to detect the antimalarial activity of OLP in the wild type (3D7), D10-expressing GFP-Atg18 parasite, and chloroquine-resistant (Dd2) parasite. OLP did not show the parasite inhibition in the routine in vitro culture of P. falciparum whereas OLP increased the antimalarial activity of artesunate. The molecular docking of autophagy-related proteins, investigations with MMI, and parasite inhibition assays indicated that the host activated the autophagy to survive OLP pressure. The challenge model of P. berghei infection showed to induce autophagy for circumventing anti-plasmodial defenses.

Theranostics: aptamer-assisted carbon nanotubes as MRI contrast and photothermal agent for breast cancer therapy.

Breast cancer is one of the leading causes of death among women globally, making its diagnosis and treatment challenging. The use of nanotechnology for cancer diagnosis and treatment is an emerging area of research. To address this issue, multiwalled carbon nanotubes (MWCNTs) were ligand exchanged with butyric acid (BA) to gain hydrophilic character. The successful functionalization was confirmed by FTIR spectroscopy. Surface morphology changes were observed using SEM, while TEM confirmed the structural integrity of the MWCNTs after functionalization. Particle size, zeta potential, and UV spectroscopy were also performed to further characterize the nanoparticles. The breast cancer aptamer specific to Mucin-1 (MUC-1) was then conjugated with the functionalized MWCNTs. These MWCNTs successfully targeted breast cancer cells (MDA-MB-231) as examined by cellular uptake studies and exhibited a reduction in cancer-induced inflammation, as evidenced by gene transcription (qPCR) and protein expression (immunoblotting) levels. Immunoblot and confocal-based immunofluorescence assay (IFA) indicated the ability of CNTs to induce photothermal cell death of MDA-MB-231 cells. Upon imaging, cancer cells were effectively visualized due to the MWCNTs' ability to act as magnetic resonance imaging (MRI) contrast agents. Additionally, MWCNTs demonstrated photothermal capabilities to eliminate bound cancer cells. Collectively, our findings pave the way for developing aptamer-labeled MWCNTs as viable "theranostic alternatives" for breast cancer treatment.

Role of natural killer and B cell interaction in inducing pathogen specific immune responses.

The innate lymphoid cell (ILC) system comprising of the circulating and tissue-resident cells is known to clear infectious pathogens, establish immune homeostasis as well as confer antitumor immunity. Human natural killer cells (hNKs) and other ILCs carry out mopping of the infectious pathogens and perform cytolytic activity regulated by the non-adaptive immune system. The NK cells generate immunological memory and rapid recall response tightly regulated by the adaptive immunity. The interaction of NK and B cell, and its role to induce the pathogen specific immunity is not fully understood. Hence, present article sheds light on the interaction between NK and B cells and resulting immune responses in the infectious diseases. The immune responses elicited by the NK-B cell interaction is of particular importance for developing therapeutic vaccines against the infectious pathogens. Further, experimental evidences suggest the immune-response driven by NK cell population elicits the host-specific antibodies and memory B cells. Also, recently developed humanized immune system (HIS) mice and their importance in to understanding the NK-B cell interaction and resulting pathogen specific immunity has been discussed.

Aceclofenac and methotrexate combination therapy could influence Th1/Th17 axis to modulate rheumatoid-arthritis-induced inflammation.

Rheumatoid arthritis (RA) is an inflammatory, autoimmune and connective-tissue arthropathy. The methotrexate (MTX) and aceclofenac (ACL) combination drug regimen is known to regulate the immunological pathways. Also, RA-elicited inflammation is decreased by the combination drug treatment. ACL and MTX combination treatment has been shown to regulate the signaling pathway controlled by NF-κB and FOXO1. The present manuscript reviews the importance of the combination drug regimen to treat and/or manage RA. The combination drug regimen could affect the Th1/Th17 axis to switch the balance toward the immunoregulatory (Th1) phenotype for establishing immune homeostasis. In conclusion, we propose the study of the immunological signaling pathways in experimental humanized RA mice.

Understanding Mutations in Human SARS-CoV-2 Spike Glycoprotein: A Systematic Review & Meta-Analysis.

Genetic variant(s) of concern (VoC) of SARS-CoV-2 have been emerging worldwide due to mutations in the gene encoding spike glycoprotein. We performed comprehensive analyses of spike protein mutations in the significant variant clade of SARS-CoV-2, using the data available on the Nextstrain server. We selected various mutations, namely, A222V, N439K, N501Y, L452R, Y453F, E484K, K417N, T478K, L981F, L212I, N856K, T547K, G496S, and Y369C for this study. These mutations were chosen based on their global entropic score, emergence, spread, transmission, and their location in the spike receptor binding domain (RBD). The relative abundance of these mutations was mapped with global mutation D614G as a reference. Our analyses suggest the rapid emergence of newer global mutations alongside D614G, as reported during the recent waves of COVID-19 in various parts of the world. These mutations could be instrumentally imperative for the transmission, infectivity, virulence, and host immune system's evasion of SARS-CoV-2. The probable impact of these mutations on vaccine effectiveness, antigenic diversity, antibody interactions, protein stability, RBD flexibility, and accessibility to human cell receptor ACE2 was studied in silico. Overall, the present study can help researchers to design the next generation of vaccines and biotherapeutics to combat COVID-19 infection.

NKB cells: A double-edged sword against inflammatory diseases.

Interferon-γ (IFN-γ)-producing natural killer (NK) cells and innate lymphoid cells (ILCs) activate the adaptive system's B and T cells in response to pathogenic invasion; however, how these cells are activated during infections is not yet fully understood. In recent years, a new lymphocyte population referred to as "natural killer-like B (NKB) cells", expressing the characteristic markers of innate NK cells and adaptive B cells, has been identified in both the spleen and mesenteric lymph nodes during infectious and inflammatory pathologies. NKB cells produce IL-18 and IL-12 cytokines during the early phases of microbial infection, differentiating them from conventional NK and B cells. Emerging evidence indicates that NKB cells play key roles in clearing microbial infections. In addition, NKB cells contribute to inflammatory responses during infectious and inflammatory diseases. Hence, the role of NKB cells in disease pathogenesis merits further study. An in-depth understanding of the phenotypic, effector, and functional properties of NKB cells may pave the way for the development of improved vaccines and therapeutics for infectious and inflammatory diseases.

Corrigendum: Nanostructured lipid carrier-mediated transdermal delivery of aceclofenac hydrogel present an effective therapeutic approach for inflammatory diseases.

[This corrects the article DOI: 10.3389/fphar.2021.713616.].