A microbially produced AhR ligand promotes a Tph1-driven tolerogenic program in multiple sclerosis.

Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases.

Impaired neutralizing antibody efficacy of tixagevimab-cilgavimab 150+150 mg as pre-exposure prophylaxis against Omicron BA.5. A real-world experience in booster vaccinated immunocompromised patients.

BACKGROUND: Tixagevimab-cilgavimab has been approved as primary pre-exposure prophylaxis in immunocompromised patients as support or replacement for vaccination, even though the Omicron variant of concern (VOC) was spreading at the time. OBJECTIVES: The aim of our study was to evaluate the post-injection neutralising activity (NT90-Abs titre) against the Omicron BA.5 variant in fully vaccinated immunocompromised patients. STUDY DESIGN: NT90-Abs titres against BA.5 and 20A.EU1 as well as anti-spike and anti-receptor-binding domain IgG were evaluated 0, 14, and 30 d after tixagevimab-cilgavimab administration. The primary end point was NT90-Abs titres ≥ 80 against BA.5 in ≥ 25% of patients, and the secondary end point was NT90-Abs titres ≥ 1280 against 20A.EU1 in >50% of patients on day 14. RESULTS: At baseline, 35.2%, 37.02%, and 32.5% of booster vaccinated patients exhibited undetectable levels of anti-S and anti-RBD IgG antibodies such as NT90-Abs titres against A20.EU1. Moreover, 35 patients (61.5%) had undetectable NT90-Abs titres against BA.5. On day 14, IgG anti-S and anti-RBD levels were 3880 BAU/mL and 776.6 AU/mL, respectively. Only 12.5% of patients met a NT90-Abs titres ≥ 80 against BA.5, whereas the median NT90-Abs titre against 20A.EU1 was 1280. NT90-Abs titres against BA.5 were 64-fold lower than those against A20.EU1. Four patients (7.5%) had a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the 3 months after treatment, all with a time gap between the booster vaccination and injection. CONCLUSIONS: To date, tixagevimab-cilgavimab cannot be considered a substitute for vaccination but may be a useful supporting therapy if the recommended dose for pre-exposure prophylaxis is doubled.

Anopheline mosquito saliva contains bacteria that are transferred to a mammalian host through blood feeding.

Introduction: Malaria transmission occurs when Plasmodium sporozoites are transferred from the salivary glands of anopheline mosquitoes to a human host through the injection of saliva. The need for better understanding, as well as novel modes of inhibiting, this key event in transmission has driven intense study of the protein and miRNA content of saliva. Until now the possibility that mosquito saliva may also contain bacteria has remained an open question despite the well documented presence of a rich microbiome in salivary glands. Methods: Using both 16S rRNA sequencing and MALDI-TOF approaches, we characterized the composition of the saliva microbiome of An. gambiae and An. stephensi mosquitoes which respectively represent two of the most important vectors for the major malaria-causing parasites P. falciparum and P. vivax. Results: To eliminate the possible detection of non-mosquito-derived bacteria, we used a transgenic, fluorescent strain of one of the identified bacteria, Serratiamarcescens, to infect mosquitoes and detect its presence in mosquito salivary glands as well as its transfer to, and colonization of, mammalian host tissues following a mosquito bite. We also showed that Plasmodium infection modified the mosquito microbiota, increasing the presence of Serratia while diminishing the presence of Elizabethkingia and that both P. berghei and Serratia were transferred to, and colonized mammalian tissues. Discussion: These data thus document the presence of bacteria in mosquito saliva, their transfer to, and growth in a mammalian host as well as possible interactions with Plasmodium transmission. Together they raise the possible role of mosquitoes as vectors of bacterial infection and the utility of commensal mosquito bacteria for the development of transmission-blocking strategies within a mammalian host.

Tripartite interactions comprising yeast-endobacteria systems in the gut of vector mosquitoes.

It is shown that bacteria use yeast as a niche for survival in stressful conditions, therefore yeasts may act as temporary or permanent bacterial reservoirs. Endobacteria colonise the fungal vacuole of various osmotolerant yeasts which survive and multiply in sugar-rich sources such as plant nectars. Nectar-associated yeasts are present even in the digestive system of insects and often establish mutualistic symbioses with both hosts. Research on insect microbial symbioses is increasing but bacterial-fungal interactions are yet unexplored. Here, we have focused on the endobacteria of Wickerhamomyces anomalus (formerly Pichia anomala and Candida pelliculosa), an osmotolerant yeast associated with sugar sources and the insect gut. Symbiotic strains of W. anomalus influence larval development and contribute digestive processes in adults, in addition to exerting wide antimicrobial properties for host defence in diverse insects including mosquitoes. Antiplasmodial effects of W. anomalus have been shown in the gut of the female malaria vector mosquito Anopheles stephensi. This discovery highlights the potential of utilizing yeast as a promising tool for symbiotic control of mosquito-borne diseases. In the present study, we have carried out a large Next Generation Sequencing (NGS) metagenomics analysis including W. anomalus strains associated with vector mosquitoes Anopheles, Aedes and Culex, which has highlighted wide and heterogeneous EB communities in yeast. Furthermore, we have disclosed a Matryoshka-like association in the gut of A stephensi that comprises different EB in the strain of W. anomalus WaF17.12. Our investigations started with the localization of fast-moving bacteria-like bodies within the yeast vacuole of WaF17.12. Additional microscopy analyses have validated the presence of alive intravacuolar bacteria and 16S rDNA libraries from WaF17.12 have identified a few bacterial targets. Some of these EB have been isolated and tested for lytic properties and capability to re-infect the yeast cell. Moreover, a selective competence to enter yeast cell has been shown comparing different bacteria. We suggested possible tripartite interactions among EB, W. anomalus and the host, opening new knowledge on the vector biology.

Characterization of lab-based swarms of Anopheles gambiae mosquitoes using 3D-video tracking.

Mosquito copulation is a crucial determinant of its capacity to transmit malaria-causing Plasmodium parasites as well as underpinning several highly-anticipated vector control methodologies such as gene drive and sterile insect technique. For the anopheline mosquitoes responsible for African malaria transmission, mating takes place within crepuscular male swarms which females enter solely to mate. However, the mechanisms that regulate swarm structure or that govern mate choice remain opaque. We used 3D-video tracking approaches and computer vision algorithms developed for the study of other complex biological systems to document swarming behavior of a lab-adapted Anopheles gambiae line in a lab-based setting. By reconstructing trajectories of individual mosquitoes lasting up to 15.88 s, in swarms containing upwards of 200 participants, we documented swarm-like behavior in both males and females. In single sex swarms, encounters between individuals were fleeting (< 0.75 s). By contrast, in mixed swarms, we were able to detect 79 'brief encounters' (> 0.75 s; < 2.5 s) and 17 longer-lived encounters (> 2.5 s). We also documented several examples of apparent male-male mating competition. These findings represent the first steps towards a more detailed and quantitative description of swarming and courtship behavior in one of the most important vectors of malaria.

Immediate reinfection with Omicron variant after clearance of a previous SARS-CoV-2 infection.

SARS-CoV-2 Omicron variant is spreading worldwide, causing unprecedented epidemic peaks due to its transmissibility and immune evasion. We searched in the archive of the Regional Microbiology Laboratory (Umbria, Italy) for immediate reinfection (i.e. infection occurring 25-60 days from primary infection) among 454,764 RT-PCR tests from 261,217 individuals. Lineage heterogeneity was assessed by S gene target failure phenomenon or whole genome sequencing. We found that BA.1 Omicron variant may cause immediate reinfection of patients just recovered from Delta infection. Immediate reinfection was not observed for any other combination of variants, including Delta over Alpha variant and BA.2 over BA.1 Omicron lineage.

Hitting the right note at the right time: Circadian control of audibility in Anopheles mosquito mating swarms is mediated by flight tones.

Mating swarms of malaria mosquitoes form every day at sunset throughout the tropical world. They typically last less than 30 minutes. Activity must thus be highly synchronized between the sexes. Moreover, males must identify the few sporadically entering females by detecting the females' faint flight tones. We show that the Anopheles circadian clock not only ensures a tight synchrony of male and female activity but also helps sharpen the males' acoustic detection system: By raising their flight tones to 1.5 times the female flight tone, males enhance the audibility of females, specifically at swarm time. Previously reported "harmonic convergence" events are only a random by-product of the mosquitoes' flight tone variance and not a signature of acoustic interaction between males and females. The flight tones of individual mosquitoes occupy narrow, partly non-overlapping frequency ranges, suggesting that the audibility of individual females varies across males.

Pharyngeal Microbial Signatures Are Predictive of the Risk of Fungal Pneumonia in Hematologic Patients.

The ability to predict invasive fungal infections (IFI) in patients with hematological malignancies is fundamental for successful therapy. Although gut dysbiosis is known to occur in hematological patients, whether airway dysbiosis also contributes to the risk of IFI has not been investigated. Nasal and oropharyngeal swabs were collected for functional microbiota characterization in 173 patients with hematological malignancies recruited in a multicenter, prospective, observational study and stratified according to the risk of developing IFI. A lower microbial richness and evenness were found in the pharyngeal microbiota of high-risk patients that were associated with a distinct taxonomic and metabolic profile. A murine model of IFI provided biologic plausibility for the finding that loss of protective anaerobes, such as Clostridiales and Bacteroidetes, along with an apparent restricted availability of tryptophan, is causally linked to the risk of IFI in hematologic patients and indicates avenues for antimicrobial stewardship and metabolic reequilibrium in IFI.

Comparison of two human organoid models of lung and intestinal inflammation reveals Toll-like receptor signalling activation and monocyte recruitment.

OBJECTIVES: The activation of immune responses in mucosal tissues is a key factor for the development and sustainment of several pathologies including infectious diseases and autoimmune diseases. However, translational research and personalised medicine struggle to advance because of the lack of suitable preclinical models that successfully mimic the complexity of human tissues without relying on in vivo mouse models. Here, we propose two in vitro human 3D tissue models, deprived of any resident leucocytes, to model mucosal tissue inflammatory processes. METHODS: We developed human 3D lung and intestinal organoids differentiated from induced pluripotent stem cells to model mucosal tissues. We then compared their response to a panel of microbial ligands and investigated their ability to attract and host human primary monocytes. RESULTS: Mature lung and intestinal organoids comprised epithelial (EpCAM+) and mesenchymal (CD73+) cells which responded to Toll-like receptor stimulation by releasing pro-inflammatory cytokines and expressing tissue inflammatory markers including MMP9, COX2 and CRP. When added to the organoid culture, primary human monocytes migrated towards the organoids and began to differentiate to an 'intermediate-like' phenotype characterised by increased levels of CD14 and CD16. CONCLUSION: We show that human mucosal organoids exhibit proper immune functions and successfully mimic an immunocompetent tissue microenvironment able to host patient-derived immune cells. Our experimental set-up provides a novel tool to tackle the complexity of immune responses in mucosal tissues which can be tailored to different human pathologies.

Sulforaphane-Loaded Ultradeformable Vesicles as A Potential Natural Nanomedicine for the Treatment of Skin Cancer Diseases.

Sulforaphane is a multi-action drug and its anticancer activity is the reason for the continuous growth of attention being paid to this drug. Sulforaphane shows an in vitro antiproliferative activity against melanoma and other skin cancer diseases. Unfortunately, this natural compound cannot be applied in free form on the skin due to its poor percutaneous permeation determined by its physico-chemical characteristics. The aim of this investigation was to evaluate ethosomes® and transfersomes® as ultradeformable vesicular carriers for the percutaneous delivery of sulforaphane to be used for the treatment of skin cancer diseases. The physico-chemical features of the ultradeformable vesicles were evaluated. Namely, ethosomes® and transfersomes® had mean sizes <400 nm and a polydispersity index close to 0. The stability studies demonstrated that the most suitable ultradeformable vesicles to be used as topical carriers of sulforaphane were ethosomes® made up of ethanol 40% (w/v) and phospholipon 90G 2% (w/v). In particular, in vitro studies of percutaneous permeation through human stratum corneum and epidermis membranes showed an increase of the percutaneous permeation of sulforaphane. The antiproliferative activity of sulforaphane-loaded ethosomes® was tested on SK-MEL 28 and improved anticancer activity was observed in comparison with the free drug.

Large-cage assessment of a transgenic sex-ratio distortion strain on populations of an African malaria vector.

BACKGROUND: Novel transgenic mosquito control methods require progressively more realistic evaluation. The goal of this study was to determine the effect of a transgene that causes a male-bias sex ratio on Anopheles gambiae target populations in large insectary cages. METHODS: Life history characteristics of Anopheles gambiae wild type and Ag(PMB)1 (aka gfp124L-2) transgenic mosquitoes, whose progeny are 95% male, were measured in order to parameterize predictive population models. Ag(PMB)1 males were then introduced at two ratios into large insectary cages containing target wild type populations with stable age distributions and densities. The predicted proportion of females and those observed in the large cages were compared. A related model was then used to predict effects of male releases on wild mosquitoes in a west African village. RESULTS: The frequency of transgenic mosquitoes in target populations reached an average of 0.44 ± 0.02 and 0.56 ± 0.02 after 6 weeks in the 1:1 and in the 3:1 release ratio treatments (transgenic male:wild male) respectively. Transgenic males caused sex-ratio distortion of 73% and 80% males in the 1:1 and 3:1 treatments, respectively. The number of eggs laid in the transgenic treatments declined as the experiment progressed, with a steeper decline in the 3:1 than in the 1:1 releases. The results of the experiment are partially consistent with predictions of the model; effect size and variability did not conform to the model in two out of three trials, effect size was over-estimated by the model and variability was greater than anticipated, possibly because of sampling effects in restocking. The model estimating the effects of hypothetical releases on the mosquito population of a West African village demonstrated that releases could significantly reduce the number of females in the wild population. The interval of releases is not expected to have a strong effect. CONCLUSIONS: The biological data produced to parameterize the model, the model itself, and the results of the experiments are components of a system to evaluate and predict the performance of transgenic mosquitoes. Together these suggest that the Ag(PMB)1 strain has the potential to be useful for reversible population suppression while this novel field develops.

Structure-activity relationships, biological evaluation and structural studies of novel pyrrolonaphthoxazepines as antitumor agents.

Microtubule-targeting agents (MTAs) are a class of clinically successful anti-cancer drugs. The emergence of multidrug resistance to MTAs imposes the need for developing new MTAs endowed with diverse mechanistic properties. Benzoxazepines were recently identified as a novel class of MTAs. These anticancer agents were thoroughly characterized for their antitumor activity, although, their exact mechanism of action remained elusive. Combining chemical, biochemical, cellular, bioinformatics and structural efforts we developed improved pyrrolonaphthoxazepines antitumor agents and their mode of action at the molecular level was elucidated. Compound 6j, one of the most potent analogues, was confirmed by X-ray as a colchicine-site MTA. A comprehensive structural investigation was performed for a complete elucidation of the structure-activity relationships. Selected pyrrolonaphthoxazepines were evaluated for their effects on cell cycle, apoptosis and differentiation in a variety of cancer cells, including multidrug resistant cell lines. Our results define compound 6j as a potentially useful optimized hit for the development of effective compounds for treating drug-resistant tumors.

Anticarcinogenic activities of sulforaphane are influenced by Nerve Growth Factor in human melanoma A375 cells.

Melanoma is a severe form of cancer, resistant to conventional therapies. According to in vitro studies, sulforaphane, a dietary component, has been considered a promising antineoplastic candidate. The present study analyzes the in vitro biological effects of sulforaphane in A375 melanoma cell line with or without the addition of Nerve Growth Factor. For the first time, our results show that a supplementation of Nerve Growth Factor partially reverses the sulforaphane-induced: i) inhibition of cell migration, ii) pro apoptotic changes in cell cycle and iii) modulation of active caspase-3. Furthermore, we report the sulforaphane-induced modulation in the expression of Nerve Growth Factor receptors TrKA and p75NTR, shifting their ratio from pro survival to pro apoptotic. In conclusion, the present study evidences that in vivo the antineoplastic effects of sulforaphane may be reduced by the contemporaneous presence of other biological elements such as Nerve Growth Factor and it contributes to a better definition of the real in vivo potentiality of sulforaphane as antineoplastic candidate.

Antitumor activity and expression profiles of genes induced by sulforaphane in human melanoma cells.

PURPOSE: Human melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistance to apoptosis, reprogramming, proliferation and survival during tumour progression. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, plays a role in carcinogenesis in many cancer types. However, the cytotoxic molecular mechanisms and gene expression profiles promoted by SFN in human melanoma remain unknown. METHODS: Three different cell lines were used: two human melanoma A375 and 501MEL and human epidermal melanocytes (HEMa). Cell viability and proliferation, cell cycle analysis, cell migration and invasion and protein expression and phosphorylation status of Akt and p53 upon SFN treatment were determined. RNA-seq of A375 was performed at different time points after SFN treatment. RESULTS: We demonstrated that SFN strongly decreased cell viability and proliferation, induced G2/M cell cycle arrest, promoted apoptosis through the activation of caspases 3, 8, 9 and hampered migration and invasion abilities in the melanoma cell lines. Remarkably, HEMa cells were not affected by SFN treatment. Transcriptomic analysis revealed regulation of genes involved in response to stress, apoptosis/cell death and metabolic processes. SFN upregulated the expression of pro-apoptotic genes, such as p53, BAX, PUMA, FAS and MDM2; promoted cell cycle inhibition and growth arrest by upregulating EGR1, GADD45B, ATF3 and CDKN1A; and simultaneously acted as a potent inhibitor of genotoxicity by launching the stress-inducible protein network (HMOX1, HSPA1A, HSPA6, SOD1). CONCLUSION: Overall, the data show that SFN cytotoxicity in melanoma derives from complex and concurrent mechanisms during carcinogenesis, which makes it a promising cancer prevention agent.

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.].

Transcriptomic profiling of microglia reveals signatures of cell activation and immune response, during experimental cerebral malaria.

Cerebral malaria is a pathology involving inflammation in the brain. There are many immune cell types activated during this process, but there is little information on the response of microglia, in this severe complication. We examined microglia by genome wide transcriptomic analysis in a model of experimental cerebral malaria (ECM), in which C57BL/6 mice are infected with Plasmodium berghei ANKA. Thousands of transcripts were differentially expressed in microglia at two different time points during infection. Proliferation of microglia was a dominant feature before the onset of ECM, and supporting this, we observed an increase in numbers of these cells in the brain. When cerebral malaria symptoms were manifest, genes involved in immune responses and chemokine production were upregulated, which were possibly driven by Type I Interferon. Consistent with this hypothesis, in vitro culture of a microglial cell line with Interferon-ß, but not infected red blood cells, resulted in production of several of the chemokines shown to be upregulated in the gene expression analysis. It appears that these responses are associated with ECM, as microglia from mice infected with a mutant P. berghei parasite (ΔDPAP3), which does not cause ECM, did not show the same level of activation or proliferation.

Release of Plasmodium sporozoites requires proteins with histone-fold dimerization domains.

The sporozoite, the stage of the malaria parasite transmitted by the mosquito, first develops for ∼2 weeks in an oocyst. Rupture of the oocyst capsule is required for release of sporozoites, which then transfer to the salivary gland where they are injected into a new host. Here we identify two parasite proteins that we call oocyst rupture proteins 1 (ORP1) and ORP2. These proteins have a histone-fold domain (HFD) that promotes heterodimer formation in the oocyst capsule at the time of rupture. Oocyst rupture is prevented in mutants lacking either protein. Mutational analysis confirms the HFD as essential for ORP1 and ORP2 function, and heterodimer formation was verified in vitro. These two proteins are potential targets for blocking transmission of the parasite in the mosquito.

Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

Comparison of Model Predictions and Laboratory Observations of Transgene Frequencies in Continuously-Breeding Mosquito Populations.

The persistence of transgenes in the environment is a consideration in risk assessments of transgenic organisms. Combining mathematical models that predict the frequency of transgenes and experimental demonstrations can validate the model predictions, or can detect significant biological deviations that were neither apparent nor included as model parameters. In order to assess the correlation between predictions and observations, models were constructed to estimate the frequency of a transgene causing male sexual sterility in simulated populations of a malaria mosquito Anopheles gambiae that were seeded with transgenic females at various proportions. Concurrently, overlapping-generation laboratory populations similar to those being modeled were initialized with various starting transgene proportions, and the subsequent proportions of transgenic individuals in populations were determined weekly until the transgene disappeared. The specific transgene being tested contained a homing endonuclease gene expressed in testes, I-PpoI, that cleaves the ribosomal DNA and results in complete male sexual sterility with no effect on female fertility. The transgene was observed to disappear more rapidly than the model predicted in all cases. The period before ovipositions that contained no transgenic progeny ranged from as little as three weeks after cage initiation to as long as 11 weeks.