Bifidobacterium and Lactobacillus Probiotics and Gut Dysbiosis in Preterm Infants: The PRIMAL Randomized Clinical Trial.

Importance: The effects of probiotic interventions on colonization with resistant bacteria and early microbiome development in preterm infants remain to be clarified. Objective: To examine the efficacy of Bifidobacterium longum subsp infantis, Bifidobacterium animalis subsp lactis (BB-12), and Lactobacillus acidophilus (La-5) probiotics to prevent colonization with multidrug-resistant organisms or highly epidemic bacteria (MDRO+) and to shape the microbiome of preterm infants toward the eubiotic state of healthy full-term infants. Design, Setting, and Participants: The multicenter, double-blinded, placebo-controlled, group sequential, phase 3 Priming Immunity at the Beginning of Life (PRIMAL) randomized clinical trial, conducted from April 2018 to June 2020, included infants with gestational age of 28 to 32 weeks at 18 German neonatal units. Data analyses were conducted from March 2020 to August 2023. Intervention: A total of 28 days of multistrain probiotics diluted in human milk/formula starting within the first 72 hours of life. Main Outcomes and Measures: Colonization with MDRO+ at day 30 of life (primary end point), late-onset sepsis and severe gastrointestinal complication (safety end points), and gut dysbiosis, ie, deviations from the microbiome of healthy, term infants (eubiosis score) based on 16-subunit ribosomal RNA and metagenomic sequencing. Results: Among the 643 infants randomized until the stop of recruitment based on interim results, 618 (median [IQR] gestational age, 31.0 [29.7-32.1] weeks; 333 male [53.9%]; mean [SD] birth weight, 1502 [369] g) had follow-up at day 30. The interim analysis with all available data from 219 infants revealed MDRO+ colonization in 43 of 115 infants (37.4%) in the probiotics group and in 39 of 104 infants (37.5%) in the control group (adjusted risk ratio, 0.99; 95% CI, 0.54-1.81; P = .97). Safety outcomes were similar in both groups, ie, late-onset sepsis (probiotics group: 8 of 316 infants [2.5%]; control group: 12 of 322 infants [3.7%]) and severe gastrointestinal complications (probiotics group: 6 of 316 infants [1.9%]; control group: 7 of 322 infants [2.2%]). The probiotics group had higher eubiosis scores than the control group at the genus level (254 vs 258 infants; median scores, 0.47 vs 0.41; odds ratio [OR], 1.07; 95% CI, 1.02-1.13) and species level (96 vs 83 infants; median scores, 0.87 vs 0.59; OR, 1.28; 95% CI, 1.19-1.38). Environmental uptake of the B infantis probiotic strain in the control group was common (41 of 84 [49%]), which was highly variable across sites and particularly occurred in infants with a sibling who was treated with probiotics. Conclusions and Relevance: Multistrain probiotics did not reduce the incidence of MDRO+ colonization at day 30 of life in preterm infants but modulated their microbiome toward eubiosis. Trial Registration: German Clinical Trials Register: DRKS00013197.

A phase 3, randomised, observer-blinded, placebo controlled-trial evaluating the safety and immunogenicity of investigational SARS-CoV-2 mRNA vaccine CVnCoV in adult healthcare workers in Mainz (Germany).

Background: CV-NCOV-005 was conducted to generate additional safety and immunogenicity data for the former CVnCoV SARS-CoV-2 mRNA vaccine candidate in healthcare workers (HCW). Methods: Randomised, observer blinded, placebo-controlled, phase 3 trial performed at the University Medical Center Mainz, Germany. HCWs aged ≥18 years with no history of SARS-CoV-2 infection/positive serology were randomly assigned to receive two doses of CVnCoV, or two doses of placebo (0.9% NaCl). The primary objectives were to expand the safety database of CVnCoV and assess antibody responses against SARS-CoV-2. Primary safety and reactogenicity outcomes included solicited adverse events (AEs) within 7 days after each dose and unsolicited AEs within 28 days after each dose, with safety follow-up for 13 months after first vaccination. Since HCWs became eligible to receive an authorised vaccine during enrolment and efficacy results from HERALD CVnCoV trial were made available on 30th of June 2021, this study was unblinded and converted to an open label design. Results: Most participants in the CVnCoV group reported at least one solicited AE, a relatively high number being Grade 3 (43.3% in CVnCoV group and 6.4% in placebo group). Most AEs were short in duration and did not affect vaccine compliance. The percentage of participants with unsolicited AEs up to 28 days after any dose was slightly higher in CVnCoV group (37.0%) compared with placebo group (31.2%). IgG binding antibodies against the receptor binding domain of the SARS-CoV-2 spike protein were observed after vaccination, with higher seroconversion rates and antibody levels after the second dose. Conclusion: No safety concerns for CVnCoV were identified up to 1 year post second dose. IgG responses against SARS-CoV-2 were observed after two doses, with a higher seroconversion rate and antibody levels observed after second vaccination.Study registration: ClinicalTrials.gov NCT04674189, study period: 23rd of December 2020 to 8th of June 2022.

Optimizing mRNA-Loaded Lipid Nanoparticles as a Potential Tool for Protein-Replacement Therapy.

Lipid nanoparticles (LNPs) tailored for mRNA delivery were optimized to serve as a platform for treating metabolic diseases. Four distinct lipid mixes (LMs) were formulated by modifying various components: LM1 (ALC-0315/DSPC/Cholesterol/ALC-0159), LM2 (ALC-0315/DOPE/Cholesterol/ALC-0159), LM3 (ALC-0315/DSPC/Cholesterol/DMG-PEG2k), and LM4 (DLin-MC3-DMA/DSPC/Cholesterol/ALC-0159). LNPs exhibited stability and homogeneity with a mean size of 75 to 90 nm, confirmed by cryo-TEM and SAXS studies. High mRNA encapsulation (95-100%) was achieved. LNPs effectively delivered EGFP-encoding mRNA to HepG2 and DC2.4 cell lines. LNPs induced cytokine secretion from human peripheral blood mononuclear cells (PBMCs), revealing that LM1, LM2, and LM4 induced 1.5- to 4-fold increases in IL-8, TNF-α, and MCP-1 levels, while LM3 showed minimal changes. Reporter mRNA expression was observed in LNP-treated PBMCs. Hemotoxicity studies confirmed formulation biocompatibility with values below 2%. In vivo biodistribution in mice post intramuscular injection showed significant mRNA expression, mainly in the liver. The modification of LNP components influenced reactogenicity, inflammatory response, and mRNA expression, offering a promising platform for selecting less reactogenic carriers suitable for repetitive dosing in metabolic disease treatment.

A prospective cohort study of SARS-CoV-2 infection-induced seroconversion and disease incidence in German healthcare workers before and during the rollout of COVID-19 vaccines.

We assessed the seroepidemiology of SARS-CoV-2 infection and the incidence of coronavirus disease 2019 (COVID-19) before and during the rollout of COVID-19 vaccines, in a prospective observational cohort study on healthcare workers (HCWs) in a large tertiary hospital in Mainz, Germany. Antibody status was assessed during six visits between September 2020 and February 2022. Self-reported symptoms were collected using a smartphone application; symptomatic HCWs were tested using real-time polymerase chain reaction (RT-PCR) assays for SARS-CoV-2. Rates of virologically confirmed and severe COVID-19 were estimated using the U.S. Food and Drug Administration (FDA) and Coalition for Epidemic Preparedness Innovations (CEPI) case definitions, respectively, and were contrasted to background community transmission and circulating SARS-CoV-2 variants. A total of 3665 HCWs were enrolled (mean follow-up time: 18 months); 97 met the FDA definition of virologically confirmed COVID-19 (incidence rate (IR) 2.3/1000 person-months (PMs), one severe case). Most cases reported ≥2 symptoms, commonly, cough and anosmia or ageusia. Overall, 263 individuals seroconverted (IR 6.6/1000 PMs-2.9 times the estimated IR of COVID-19), indicating many cases were missed, either due to asymptomatic infections or to an atypical presentation of symptoms. A triphasic trend in anti-SARS-CoV-2 seroprevalence and seroconversion was observed, with an initial increase following the rollout of COVID-19 vaccines, a two-fold decline six months later, and finally a six-fold increase by the end of the study when Omicron was the dominant circulating variant. Despite the increase in infection rates at the end of the study due to the circulation of the Omicron variant, the infection and disease rates observed were lower than the published estimates in HCWs and rates in the general local population. Preferential vaccination of HCWs and the strict monitoring program for SARS-CoV-2 infection are the most likely reasons for the successful control of COVID-19 in this high-risk population.

Challenges in Diagnosing and Treating Acutely Febrile Children with Suspected Malaria at Health Care Facilities in the Lake Mwanza Region of Tanzania.

Acute febrile diseases transmitted by mosquitos are a diagnostic challenge for pediatricians working in sub-Saharan Africa. Misclassification due to the lack of rapid, reliable diagnostic tests leads to the overuse of antibiotics and antimalarials. Children presenting with acute fever and suspected of having malaria were examined at health care facilities in the Mwanza Region of Tanzania. The sensitivity and specificity of blood smear microscopy and malaria rapid diagnostic tests that targeted histidine-rich protein 2 and Plasmodium lactate dehydrogenase were compared with a multiplex reverse transcriptase-polymerase chain reaction (PCR)-ELISA. Six hundred ninety-eight children presented with acute fever and met the criteria for inclusion; 23% received antibiotics and 23% received antimalarials prior to admission. Subsequently, 20% were confirmed by PCR to have Plasmodium falciparum infection. Blood smear microscopy exhibited 33% sensitivity and 93% specificity. The malaria rapid test provided 87% sensitivity and 98% specificity in detecting acute malaria infections. Only 7% of malaria-negative children received antimalarials at Sengerema Designated District Hospital when treatment was guided by the results of rapid testing. In contrast, 75% of malaria-negative patients were treated with antimalarial drugs at health facilities that used blood smears as the standard diagnostic test. Misclassification and premedication of nonmalarial, febrile illnesses contribute to the emergence of antimalarial and antimicrobial resistance. The incorporation of malaria rapid diagnostic tests into the clinical routine translated into improved treatment and a significant reduction in antimalarial drug prescriptions.

Comparison of Inflammatory Cytokine Levels in Hepatic and Jugular Veins of Patients with Cirrhosis.

Background: Systemic inflammation with elevated inflammatory cytokines is a hallmark in patients with cirrhosis and the main driver of decompensation. There is insufficient data on whether inflammatory cytokine levels differ between hepatic and jugular veins, which may have implications for further immunological studies. Methods: Blood from the hepatic and jugular veins of 40 patients with cirrhosis was collected during hepatic venous pressure gradient (HVPG) measurements. Serum levels of 13 inflammatory cytokines (IL-1ß, Int-α2, Int-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33) were quantified by cytometric bead array. Results: Cytokine levels of IFN-α2, IFN-γ, TNF-α, IL-6, IL-8, IL-10, IL-17A, IL-18, IL-23, and IL-33 were significantly elevated in patients with decompensated cirrhosis compared to patients with compensated cirrhosis. When comparing patients with clinically significant portal hypertension (CSPH, HVPG ≥ 10 mmHg) to patients without CSPH, there were significantly enhanced serum levels of IL-6 and IL-18 in the former group. There was no significant difference between cytokine serum levels between blood obtained from the jugular versus hepatic veins. Even in subgroup analyses stratified for an early cirrhosis stage (Child-Pugh (CP) A) or more decompensated stages (CP B/C), cytokine levels were similar. Conclusion: Cytokine levels increase with decompensation and increasing portal hypertension in patients with cirrhosis. There is no relevant difference in cytokine levels between hepatic and jugular blood in patients with cirrhosis.

Multiplex Reverse Transcription Polymerase Chain Reaction Combined with a Microwell Hybridization Assay Screening for Arbovirus and Parasitic Infections in Febrile Patients Living in Endemic Regions of Colombia.

Acute febrile syndrome is a frequent reason for medical consultations in tropical and subtropical countries where the cause could have an infectious origin. Malaria and dengue are the primary etiologies in Colombia. As such, constant epidemiological surveillance and new diagnostic tools are required to identify the causative agents. A descriptive cross-sectional study was conducted to evaluate the circulation and differential diagnosis of six pathogens in two regions of Colombia. The results obtained via multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) were comparable to those obtained using rapid tests conducted at the time of patient enrollment. Of 155 patients evaluated, 25 (16.1%) and 16 (10.3%) were positive for malaria and dengue, respectively; no samples were positive for any of the other infectious agents tested. In most cases, m-RT-PCR-ELISA confirmed the results previously obtained through rapid testing.

Seroprevalence of Dengue and Chikungunya Virus Infections in Children Living in Sub-Saharan Africa: Systematic Review and Meta-Analysis.

Dengue and chikungunya viruses are frequent causes of malarial-like febrile illness in children. The rapid increase in virus transmission by mosquitoes is a global health concern. This is the first systematic review and meta-analysis of the childhood prevalence of dengue and chikungunya in Sub-Saharan Africa (SSA). A comprehensive search of the MEDLINE (Ovid), Embase (Ovid), and Cochrane Library (Wiley) databases was conducted on 28 June 2019, and updated on 12 February 2022. The search strategy was designed to retrieve all articles pertaining to arboviruses in SSA children using both controlled vocabulary and keywords. The pooled (weighted) proportion of dengue and chikungunya was estimated using a random effect model. The overall pooled prevalence of dengue and chikungunya in SSA children was estimated to be 16% and 7%, respectively. Prevalence was slightly lower during the period 2010-2020 compared to 2000-2009. The study design varied depending on the healthcare facility reporting the disease outbreak. Importantly, laboratory methods used to detect arbovirus infections differed. The present review documents the prevalence of dengue and chikungunya in pediatric patients throughout SSA. The results provide unprecedented insight into the transmission of dengue and chikungunya viruses among these children and highlight the need for enhanced surveillance and controlled methodology.

Cytotoxic Screening and Enhanced Anticancer Activity of Lippia alba and Clinopodium nepeta Essential Oils-Loaded Biocompatible Lipid Nanoparticles against Lung and Colon Cancer Cells.

Plant and herbal essential oils (EOs) offer a wide range of pharmacological actions that include anticancer effects. Here, we evaluated the cytotoxic activity of EO from Lippia alba (chemotype linalool), L. alba (chemotype dihydrocarvone, LaDEO), Clinopodium nepeta (L.) Kuntze (CnEO), Eucalyptus globulus, Origanum × paniculatum, Mentha × piperita, Mentha arvensis L., and Rosmarinus officinalis L. against human lung (A549) and colon (HCT-116) cancer cells. The cells were treated with increasing EO concentrations (0-500 µL/L) for 24 h, and cytotoxic activity was assessed. LaDEO and CnEO were the most potent EOs evaluated (IC50 range, 145-275 µL/L). The gas chromatography-mass spectrometry method was used to determine their composition. Considering EO limitations as therapeutic agents (poor water solubility, volatilization, and oxidation), we evaluated whether LaDEO and CnEO encapsulation into solid lipid nanoparticles (SLN/EO) enhanced their anticancer activity. Highly stable spherical SLN/LaDEO and SLN/CnEO SLN/EO were obtained, with a mean diameter of 140-150 nm, narrow size dispersion, and Z potential around -5mV. EO encapsulation strongly increased their anticancer activity, particularly in A549 cells exposed to SLN/CnEO (IC50 = 66 µL/L CnEO). The physicochemical characterization, biosafety, and anticancer mechanisms of SLN/CnEO were also evaluated in A549 cells. SLN/CnEO containing 97 ± 1% CnEO was highly stable for up to 6 months. An increased in vitro CnEO release from SLN at an acidic pH (endolysosomal compartment) was observed. SLN/CnEO proved to be safe against blood components and non-toxic for normal WI-38 cells at therapeutic concentrations. SLN/CnEO substantially enhanced A549 cell death and cell migration inhibition compared with free CnEO.

Liver Cell Type-Specific Targeting by Nanoformulations for Therapeutic Applications.

Hepatocytes exert pivotal roles in metabolism, protein synthesis and detoxification. Non-parenchymal liver cells (NPCs), largely comprising macrophages, dendritic cells, hepatic stellate cells and liver sinusoidal cells (LSECs), serve to induce immunological tolerance. Therefore, the liver is an important target for therapeutic approaches, in case of both (inflammatory) metabolic diseases and immunological disorders. This review aims to summarize current preclinical nanodrug-based approaches for the treatment of liver disorders. So far, nano-vaccines that aim to induce hepatitis virus-specific immune responses and nanoformulated adjuvants to overcome the default tolerogenic state of liver NPCs for the treatment of chronic hepatitis have been tested. Moreover, liver cancer may be treated using nanodrugs which specifically target and kill tumor cells. Alternatively, nanodrugs may target and reprogram or deplete immunosuppressive cells of the tumor microenvironment, such as tumor-associated macrophages. Here, combination therapies have been demonstrated to yield synergistic effects. In the case of autoimmune hepatitis and other inflammatory liver diseases, anti-inflammatory agents can be encapsulated into nanoparticles to dampen inflammatory processes specifically in the liver. Finally, the tolerance-promoting activity especially of LSECs has been exploited to induce antigen-specific tolerance for the treatment of allergic and autoimmune diseases.

Resilience and stability of the CF- intestinal and respiratory microbiome during nutritional and exercise intervention.

BACKGROUND: Impaired respiratory and intestinal microbiome composition is linked to cystic fibrosis lung disease severity. In people with cystic fibrosis (pwCF), regular exercise is recommended to delay disease progression and preserve a stable lung function. An optimal nutritional status is vital for best clinical outcomes. Our study investigated whether regular and monitored exercise and nutritional support promotes CF microbiome health. METHODS: A personalized nutrition and exercise program promoted nutritional intake and physical fitness in 18 pwCF for 12 months. Throughout the study, patients performed strength and endurance training monitored by a sports scientist via an internet platform. After three months, food supplementation with Lactobacillus rhamnosus LGG was introduced. Nutritional status and physical fitness were assessed before the study started, after three and nine months. Sputum and stool were collected, and microbial composition was analyzed by 16S rRNA gene sequencing. RESULTS: Sputum and stool microbiome composition remained stable and highly specific to each patient during the study period. Disease-associated pathogens dominated sputum composition. Lung disease severity and recent antibiotic treatment had the highest impact on taxonomic composition in stool and sputum microbiome. Strikingly, the long-term antibiotic treatment burden had only a minor influence. CONCLUSION: Despite the exercise and nutritional intervention, respiratory and intestinal microbiomes proved to be resilient. Dominant pathogens drove the composition and functionality of the microbiome. Further studies are required to understand which therapy could destabilize the dominant disease-associated microbial composition of pwCF.

Modification of Regulatory T Cell Epitopes Promotes Effector T Cell Responses to Aspartyl/Asparaginyl ß-Hydroxylase.

Cancer is a leading cause of death worldwide. The search for innovative therapeutic approaches is a principal focus of medical research. Vaccine strategies targeting a number of tumor-associated antigens are currently being evaluated. To date, none have garnered significant success. Purportedly, an immunosuppressive tumor microenvironment and the accumulation of regulatory T cells contribute to a lack of tumor vaccine efficacy. Aspartyl/asparaginyl ß-hydroxylase (ASPH), a promising therapeutic target, is overexpressed in a variety of malignant tumors but is expressed negligibly in normal tissues. Computer analysis predicted that ASPH expresses four peptide sequences (epitopes) capable of stimulating regulatory T cell activity. The abolition of these putative regulatory T cell epitopes increased the CD4+ and CD8+ effector T cell responses to monocyte-derived dendritic cells pulsed with a modified, epitope-depleted version of ASPH in an ex vivo human lymphoid tissue-equivalent coculture system while simultaneously decreasing the overall number of FoxP3+ regulatory T cells. These findings suggest that the efficacy of all new vaccine candidates would profit from screening and eliminating potential tolerogenic regulatory T cell epitopes.

Phenylalanine hydroxylase mRNA rescues the phenylketonuria phenotype in mice.

Phenylketonuria (PKU) is an inborn error of metabolism caused by a deficiency in functional phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine (Phe) in patients' blood and organs. Affected patients encounter severe developmental delay, neurological deficits, and behavioral abnormalities when not treated. Early diagnosis and treatment are extremely important; newborn screening programs have been implemented in most countries to ensure early identification of patients with PKU. Despite available treatment options, several challenges remain: life-long adherence to a strict diet, approval of some medications for adults only, and lack of response to these therapies in a subpopulation of patients. Therefore, there is an urgent need for treatment alternatives. An mRNA-based approach tested in PKU mice showed a fast reduction in the accumulation of Phe in serum, liver and brain, the most significant organ affected. Repeated injections of LNP-formulated mouse PAH mRNA rescued PKU mice from the disease phenotype for a prolonged period of time. An mRNA-based approach could improve the quality of life tremendously in PKU patients of all ages by replacing standard-of-care treatments.

mRNA-Based Approaches to Treating Liver Diseases.

Diseases that affect the liver account for approximately 2 million deaths worldwide each year. The increasing prevalence of these diseases and the limited efficacy of current treatments are expected to stimulate substantial growth in the global market for therapeutics that target the liver. Currently, liver transplantation is the only curative option available for many liver diseases. Gene therapy represents a valuable approach to treatment. The liver plays a central role in a myriad of essential metabolic functions, making it an attractive organ for gene therapy; hepatocytes comprise the most relevant target. To date, viral vectors constitute the preferred approach to targeting hepatocytes with genes of therapeutic interest. Alternatively, mRNA-based therapy offers a number of comparative advantages. Clinical and preclinical studies undertaken to treat inherited metabolic diseases affecting the liver, cirrhosis and fibrosis, hepatocellular carcinoma, hepatitis B, and cytomegalovirus using lipid nanoparticle-encapsulated mRNAs that encode the therapeutic or antigenic protein of interest are discussed.

Colloidal delivery of vitamin E into solid lipid nanoparticles as a potential complement for the adverse effects of anemia treatment.

Vitamin E (VitE) is one of the most important antioxidants and plays a key role in decreasing the inflammatory effects of oxidative stress caused by recurrent doses of iron administration in anemia treatment. However, VitE is poorly soluble in aqueous environments. Here, VitE encapsulation into solid lipid nanoparticles (SLN) composed of myristil myristate to improve its bioavailability was proposed. A 99.9 ± 0.1% encapsulation efficiency with a drug/lipid ratio of 500 µg/mg and 478 higher VitE solubility was obtained. The antioxidant properties of VitE after encapsulation were maintained. SLN-VitE showed a 228.2 nm mean diameter with low polidispersitivity (0.335), and negative Z potential (ζ ≈ -9.0 mV). The SLN were well-dispersed, displayed spherical and homogeneous morphology by TEM. A controlled release of VitE from SLN was found. The XRD and FTIR analyses revealed the presence of a nanostructured architecture of SLN after VitE incorporation. We probed the safety of SLN-VitE after contact with three in vitro cell models: erythrocytes, lymphocytes and HepG2 cells. The cell viability in presence of SLN, SLN-VitE, and their combinations with iron was not affected. The comet assay demonstrated that the DNA damage caused by iron administration was decrease in presence of SLN-VitE.

OVA-PEG-R848 nanocapsules stimulate neonatal conventional and plasmacytoid dendritic cells.

Childhood mortality represents a major issue with 5. 3 million worldwide deaths of children under 5 years of age in 2019. Approximately half of those deaths can be attributed to easily preventable, infectious diseases. Currently approved neonatal vaccines are typically effective only after multiple doses leaving infants especially vulnerable during the first 6 months of life. Survival rates could be improved significantly by developing new and more potent vaccines that are capable of overcoming inherently tolerogenic neonatal immune systems. TLR agonists have garnered a great deal of attention in recent years due to their extensive capacities to activate innate immunity. Herein, the superior capacity of the TLR7/8 agonist, resiquimod (R848), to activate adult and neonatal primary peripheral blood dendritic cells is demonstrated. Moreover, R848 can be conjugated to polyethylene glycol and encapsulated in ovalbumin nanocapsules to efficiently co-deliver antigen and adjuvant in vitro. This study is among the first to demonstrate the capacity of encapsulated R848 to activate neonatal dendritic cells. These findings support the potential incorporation of R848 as adjuvant in neonatal vaccines, making them more effective in eliciting a robust immune response.

Nanostructured Lipid Carriers Loaded with Dexamethasone Prevent Inflammatory Responses in Primary Non-Parenchymal Liver Cells.

Liver inflammation represents a major clinical problem in a wide range of pathologies. Among the strategies to prevent liver failure, dexamethasone (DXM) has been widely used to suppress inflammatory responses. The use of nanocarriers for encapsulation and sustained release of glucocorticoids to liver cells could provide a solution to prevent severe side effects associated with systemic delivery as the conventional treatment regime. Here we describe a nanostructured lipid carrier developed to efficiently encapsulate and release DXM. This nano-formulation proved to be stable over time, did not interact in vitro with plasma opsonins, and was well tolerated by primary non-parenchymal liver cells (NPCs). Released DXM preserved its pharmacological activity, as evidenced by inducing robust anti-inflammatory responses in NPCs. Taken together, nanostructured lipid carriers may constitute a reliable platform for the delivery of DXM to treat pathologies associated with chronic liver inflammation.

mRNA-based therapy proves superior to the standard of care for treating hereditary tyrosinemia 1 in a mouse model.

Hereditary tyrosinemia type 1 is an inborn error of amino acid metabolism characterized by deficiency of fumarylacetoacetate hydrolase (FAH). Only limited treatment options (e.g., oral nitisinone) are available. Patients must adhere to a strict diet and face a life-long risk of complications, including liver cancer and progressive neurocognitive decline. There is a tremendous need for innovative therapies that standardize metabolite levels and promise normal development. Here, we describe an mRNA-based therapeutic approach that rescues Fah-deficient mice, a well-established tyrosinemia model. Repeated intravenous or intramuscular administration of lipid nanoparticle-formulated human FAH mRNA resulted in FAH protein synthesis in deficient mouse livers, stabilized body weight, normalized pathologic increases in metabolites after nitisinone withdrawal, and prevented early death. Dose reduction and extended injection intervals proved therapeutically effective. These results provide proof of concept for an mRNA-based therapeutic approach to treating hereditary tyrosinemia type 1 that is superior to the standard of care.

Enrichment Methods for Murine Liver Non-Parenchymal Cells Differentially Affect Their Immunophenotype and Responsiveness towards Stimulation.

Hepatocytes comprise the majority of the liver and largely exert metabolic functions, whereas non-parenchymal cells (NPCs)-comprising Kupffer cells, dendritic cells and liver sinusoidal endothelial cells-control the immunological state within this organ. Here, we compared the suitability of two isolation methods for murine liver NPCs. Liver perfusion (LP) with collagenase/DNase I applied via the portal vein leads to efficient liver digestion, whereas the modified liver dissociation (LD) method combines mechanical dissociation of the retrieved organ with enzymatic degradation of the extracellular matrix. In cases of both LP and LD, NPCs were enriched by subsequent gradient density centrifugation. Our results indicate that LP and LD are largely comparable with regards to the yield, purity, and composition of liver NPCs. However, LD-enriched liver NPCs displayed a higher degree of activation after overnight cultivation, and accordingly were less responsive towards stimulation with toll-like receptor ligands that are frequently used as adjuvants, e.g., in nano-vaccines. We conclude that LP is more suitable for obtaining liver NPCs for subsequent in vitro studies, whereas LD as the less laborious method, is more convenient for parallel isolation of larger numbers of samples for ex vivo analysis.

Long-Term, CD4+ Memory T Cell Response to SARS-CoV-2.

The first cases of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were reported by Chinese authorities at the end of 2019. The disease spread quickly and was declared a global pandemic shortly thereafter. To respond effectively to infection and prevent viral spread, it is important to delineate the factors that affect protective immunity. Herein, a cohort of convalescent healthcare workers was recruited and their immune responses were studied over a period of 3 to 9 months following the onset of symptoms. A cross-reactive T cell response to SARS-CoV-2 and endemic coronaviruses, i.e., OC43 and NL63, was demonstrated in the infected, convalescent cohort, as well as a cohort composed of unexposed individuals. The convalescent cohort, however, displayed an increased number of SARS-CoV-2-specific CD4+ T cells relative to the unexposed group. Moreover, unlike humoral immunity and quickly decreasing antibody titers, T cell immunity in convalescent individuals was maintained and stable throughout the study period. This study also suggests that, based on the higher CD4 T cell memory response against nucleocapsid antigen, future vaccine designs may include nucleocapsid as an additional antigen along with the spike protein.