Electroacupuncture attenuates ac4C modification of P16 mRNA in the ovarian granulosa cells of a mouse model premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) is a type of pathological aging, which seriously interferes with the fertility of affected women. Electroacupuncture (EA) may have a beneficial effect; however, its mechanism of action is unknown. The purpose of this study was to determine the effect of EA on ovarian function in ovarian granulosa cells (OGCs) in a cyclophosphamide (CTX)-induced mouse model of POF. METHODS: Mice were divided into three groups: wild type (WT) group, CTX group and CTX + EA group. EA was administered under isoflurane anesthesia at CV4, ST36 and SP6 for 30 min every 2 days, 2-3 times per week for a total of 4 weeks. Effects of EA on ovarian weight and level of estrogen were examined. The mRNA and protein expression levels of cell cycle-associated proteins were detected and mRNA modifications were analyzed. RESULTS: EA significantly increased ovarian weight and reduced the proportion of atretic follicles in mice with CTX-induced POF (p < 0.05). EA increased the level of estrogen in the peripheral blood of mice and inhibited the modification of total mRNA N4-acetylcytidine (ac4C). A significant increase in the expression of P16 and N-acetyltransferase 10 (NAT10) and a significant decrease in the expression of Cyclin D (CCND1) and cyclin-dependent kinase 6 (CDK6) were observed in the OGCs of POF mice (p<0.05). After EA, P16 and NAT10 expression was decreased, and CCND1 and CDK6 expression was increased. Finally, EA reduced the ac4C modification of P16 mRNA-specific sites in the OGCs of POF mice. CONCLUSION: This study demonstrated that EA promoted the repair of the ovarian microenvironment by inhibiting the ac4C modification of P16 mRNA to decrease its stability and expression intensity, and by altering the activity of the P16/CDK6/CCND1 axis in OGCs.

Anti-inflammatory effects of amarogentin on 2,4-dinitrochlorobenzene-induced atopic dermatitis-like mice and in HaCat cells.

BACKGROUND: Amarogentin (AMA) is a secoiridoid glycoside extracted from Swertia and Gentiana roots and exhibits many biological effects such as antioxidative, anti-inflammatory, and antitumor activities. Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by disorders in the regulation of multiple inflammatory cytokines. No effective cure has been found for AD now. METHODS: We constructed the HaCat and splenocyte model and tested the inhibitory effect of AMA on IL-4, IL-6, and IL-13 secretions using enzyme-linked immunosorbent assay (ELISA). The AD mouse model was constructed and treated with AMA, the severity of skin lesions was observed, epidermal tissue was collected, and epidermal thickness and mast cell infiltration were observed using hematoxylin and eosin and toluidine blue staining, respectively. The expression of kallikrein-related peptidase 7 (KLK7) and filaggrin (FLG) was detected using immunostaining and Western blot analysis. The mRNA expression of KLK7 and FLG was detected using quantitative polymerase chain reaction (qPCR). Blood immunoglobulin E (IgE) secretion was detected. RESULTS: AMA inhibited IL-6 secreted by tumor necrosis factor (TNF)-α-induced HaCaT cells and reduced IL-4 and IL-13 secreted by phytohemagglutinin (PHA)-induced primary cells in the mice spleen. It was found that the treatment of AMA with 2,4-dinitrochlorobenzene-induced AD-like mice could promote the recovery of dermatitis, reduce the score of dermatitis severity and the scratching frequency, treat the skin lesions, reduce the epidermal thickness, decrease the infiltration of mast cells, reduce the IgE level in serum, decrease the expression levels of AD-related cytokines, increase protein and mRNA expression of FLG, and reduce the protein and mRNA expression of KLK7 in the skin tissues of AD-like mice. CONCLUSION: In conclusion, AMA inhibits inflammatory response at the cellular level, and AMA reduces the validation response of specific dermatitis mice, relieves pruritus, and repairs the damaged skin barrier.

Time Course of Splenic Cytokine mRNA and Hormones during a Lipopolysaccharide-Induced Inflammation in Toads.

Inflammation comprises alterations in glucocorticoids (in amphibians, corticosterone-CORT) and melatonin (MEL) levels, two hormones with immunomodulatory effects on cytokine production in several vertebrates. Cytokines mediate inflammation progress differently depending on their function. While some are secreted during the acute phase of the immune response, others prevail during the resolution phase. Major efforts have been made to understand the interaction of endocrine mediators and cytokine production in endotherms, but little is known for ectotherms so far. Characterizing the stages of inflammation and their interplay with endocrine mediators is crucial for an assertive and integrative approach to amphibian physiology and ecoimmunology. Herein, we investigated CORT and MEL plasma levels as well as splenic cytokine (IL-1ß, IL-6, and IL-10) mRNA levels during the progression of the inflammatory response in toads (Rhinella diptycha) in four time-points (1, 3, 6, and 18 h) after an immune challenge with lipopolysaccharide (LPS) using independent samples. Toads were responsive to LPS, with all hormones and cytokines affected by LPS. IL-1ß and IL-6 were up-regulated after 1 h, but IL-1ß decreased right after 3 h, while IL-6 sustained up-regulation throughout all time-points. IL-10 had not been detected until 6 h post-LPS-stimulation, when it showed up-regulation, along with a CORT increase at the same time-point. After 18 h, CORT levels were still high, and IL-1ß was up-regulated again, along with up-regulated IL-6 and an IL-10 decrease. We also found positive correlations between IL-1ß with IL-6 for LPS and saline groups. LPS-treated individuals showed an overall decrease in MEL plasma levels compared to saline counterparts. Our results showcase the early endocrine and molecular events of the amphibian immune response. We also report activation of the hypothalamus-pituitary-interrenal (HPI) axis during inflammation and increasing evidence for an immune-pineal axis to be described in amphibians.

Implications of mRNA-based SARS-CoV-2 vaccination for cancer patients.

SARS-CoV-2 infection and the resulting COVID-19 have afflicted millions of people in an ongoing worldwide pandemic. Safe and effective vaccination is needed urgently to protect not only the general population but also vulnerable subjects such as patients with cancer. Currently approved mRNA-based SARS-CoV-2 vaccines seem suitable for patients with cancer based on their mode of action, efficacy, and favorable safety profile reported in the general population. Here, we provide an overview of mRNA-based vaccines including their safety and efficacy. Extrapolating from insights gained from a different preventable viral infection, we review existing data on immunity against influenza A and B vaccines in patients with cancer. Finally, we discuss COVID-19 vaccination in light of the challenges specific to patients with cancer, such as factors that may hinder protective SARS-CoV-2 immune responses in the context of compromised immunity and the use of immune-suppressive or immune-modulating drugs.

COVID-19 Vaccine Concerns: Fact or Fiction?

One year has elapsed since a team of Chinese scientists reported the first case of COVID-19 in Wuhan, China on January 8, 2020, after sequencing the first viral genetic material. Since then, many vaccines were rushed into testing, bypassing animal experimentations, with more than 200 pharma companies in different countries declaring the development of different vaccines, each with their own strategy for generating immunity, despite the arguments of many infectious disease experts that 18 months for a first vaccine is an incredibly aggressive schedule because it takes an average of 10 years to develop a vaccine. Ten vaccine candidates have already entered phase 3 clinical trials in humans. These vaccines rely on different types of technology, the most innovative of which use the genetic material messenger RNA. Many provocative questions and genuine concerns have been raised, such as short durations of efficacy and safety follow-ups, lack of identified correlates of protection, morbidity and mortality cases reported shortly after vaccination, uncertainties regarding the risk of enhanced disease on exposure to the virus in the long-term, the possibility of viral transmission after vaccination, the reported reduced efficacies of these vaccines against new variants, the efficacy and safety of these vaccines in the previously excluded subgroups (such as children, pregnant women, the frail elderly high-risk population, and immunocompromised individuals), the unknown risk of immunogenicity-induced autoimmune diseases, cancer and chronic inflammation, the risk of genome transformation (mainly in the presence of reverse transcriptase), and finally the potential coercion that may be imposed by either public or private sectors on citizens to receive the vaccine. Many plausible questions are apparent, with no clear and convincing answers.

Safe and effective aerosolization of in vitro transcribed mRNA to the respiratory tract epithelium of horses without a transfection agent.

Vaccines and therapeutics using in vitro transcribed mRNA hold enormous potential for human and veterinary medicine. Transfection agents are widely considered to be necessary to protect mRNA and enhance transfection, but they add expense and raise concerns regarding quality control and safety. We found that such complex mRNA delivery systems can be avoided when transfecting epithelial cells by aerosolizing the mRNA into micron-sized droplets. In an equine in vivo model, we demonstrated that the translation of mRNA into a functional protein did not depend on the addition of a polyethylenimine (PEI)-derived transfection agent. We were able to safely and effectively transfect the bronchial epithelium of foals using naked mRNA (i.e., mRNA formulated in a sodium citrate buffer without a delivery vehicle). Endoscopic examination of the bronchial tree and histology of mucosal biopsies indicated no gross or microscopic adverse effects of the transfection. Our data suggest that mRNA administered by an atomization device eliminates the need for chemical transfection agents, which can reduce the cost and the safety risks of delivering mRNA to the respiratory tract of animals and humans.

Modified mRNA as a Therapeutic Tool for the Heart.

Despite various clinical modalities available for patients, heart disease remains among the leading causes of mortality and morbidity worldwide. Genetic medicine, particularly mRNA, has broad potential as a therapeutic. More specifically, mRNA-based protein delivery has been used in the fields of cancer and vaccination, but recent changes to the structural composition of mRNA have led the scientific community to swiftly embrace it as a new drug to deliver missing genes to injured myocardium and many other organs. Modified mRNA (modRNA)-based gene delivery features transient but potent protein translation and low immunogenicity, with minimal risk of insertional mutagenesis. In this review, we compared and listed the advantages of modRNA over traditional vectors for cardiac therapy, with particular focus on using modRNA therapy in cardiac repair. We present a comprehensive overview of modRNA's role in cardiomyocyte (CM) proliferation, cardiac vascularization, and prevention of cardiac apoptosis. We also emphasize recent advances in modRNA delivery strategies and discuss the challenges for its clinical translation.

Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes.

Chemically modified mRNA is an efficient, biocompatible modality for therapeutic protein expression. We report a first-time-in-human study of this modality, aiming to evaluate safety and potential therapeutic effects. Men with type 2 diabetes mellitus (T2DM) received intradermal injections of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) or buffered saline placebo (ethical obligations precluded use of a non-translatable mRNA control) at randomized sites on the forearm. The only causally treatment-related adverse events were mild injection-site reactions. Skin microdialysis revealed elevated VEGF-A protein levels at mRNA-treated sites versus placebo-treated sites from about 4-24 hours post-administration. Enhancements in basal skin blood flow at 4 hours and 7 days post-administration were detected using laser Doppler fluximetry and imaging. Intradermal VEGF-A mRNA was well tolerated and led to local functional VEGF-A protein expression and transient skin blood flow enhancement in men with T2DM. VEGF-A mRNA may have therapeutic potential for regenerative angiogenesis.

Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium.

Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g-1 of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium.

Phase Ib study evaluating a self-adjuvanted mRNA cancer vaccine (RNActive®) combined with local radiation as consolidation and maintenance treatment for patients with stage IV non-small cell lung cancer.

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) represents a significant unmet medical need. Despite advances with targeted therapies in a small subset of patients, fewer than 20% of patients survive for more than two years after diagnosis. Cancer vaccines are a promising therapeutic approach that offers the potential for durable responses through the engagement of the patient's own immune system. CV9202 is a self-adjuvanting mRNA vaccine that targets six antigens commonly expressed in NSCLC (NY-ESO-1, MAGEC1, MAGEC2, 5 T4, survivin, and MUC1). METHODS/DESIGN: The trial will assess the safety and tolerability of CV9202 vaccination combined with local radiation designed to enhance immune responses and will include patients with stage IV NSCLC and a response or stable disease after first-line chemotherapy or therapy with an EGFR tyrosine kinase inhibitor. Three histological and molecular subtypes of NSCLC will be investigated (squamous and non-squamous cell with/without EGFR mutations). All patients will receive two initial vaccinations with CV9202 prior to local radiotherapy (5 GY per day for four successive days) followed by further vaccinations until disease progression. The primary endpoint of the study is the number of patients experiencing Grade >3 treatment-related adverse events. Pharmacodynamic analyses include the assessment of immune responses to the antigens encoded by CV9202 and others not included in the panel (antigen spreading) and standard efficacy assessments. DISCUSSION: RNActive self-adjuvanted mRNA vaccines offer the potential for simultaneously inducing immune responses to a wide panel of antigens commonly expressed in tumors. This trial will assess the feasibility of this approach in combination with local radiotherapy in NSCLC patients. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01915524/EudraCT No.: 2012-004230-41.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis.

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Insulin and phorbol ester regulation of gene 33 expression in CHO cells

Rat gene 33 (g33) mRNA has a widespread tissue distribution. Insulin and various agents such as glucocorticoids, phorbol esters and plant lectins regulate G33 expression in rat hepatoma cells. The regulation of g33 by insulin and a phorbol ester was examined in two Chinese Hamster ovary (CHO) cell lines, CHO-T cells (which overexpress human insulin receptors (hIR)) and wild type CHOwt cells. These cell lines were used to determine how expression of the hIR influences the capacity of g33 to respond to insulin and phorbol myristate acetate (PMA). Treatment of CHOwt and CHO-T cells with insulin increased mRNAg33 levels three to four-fold, with a maximum effect reached after three hours of treatment. PMA treatment of CHOwt and CHO-T cells caused a similar elevation of mRNAg33 levels after three hours. Insulin had no effect on mRNAg33 stability in both CHO cell lines. Additionally, the effects of insulin and PMA on mRNAg33 levels were additive only in CHO-T cells. Insulin or PMA-pretreated CHO-T cells were able to respond to both agents, but elevation ofmRNAg33 levels was maximal. In contrast, when insulin and/or PMA-pretreated CHOwt cells were exposed to insulin or PMA, g33 was able to respond maximally. These results suggest that insulin and phorbol esters act through different signaling mechanisms in CHOwt cells. Additionally, insulin's ability to stimulate g33 expression in CHOwt cells suggests that this insulin effect may be independent of the insulin eceptor. There are differences in the regulation pattern of g33 by insulin and PMA in rat hepatoma and among the two CHO cell lines used in this study