Suchergebnisse

Abstract
Schistosomiasis, a widespread neglected tropical disease, presents a complex and multifaceted clinical-pathological profile. Using hamsters as final hosts, we dissected molecular events following Schistosoma mansoni infection in the liver—the organ most severely affected in schistosomiasis patients. Employing tandem mass tag–based proteomics, we studied alterations in the liver proteins in response to various infection modes and genders. We examined livers from female and male hamsters that were: noninfected (control), infected with either unisexual S. mansoni cercariae (single-sex) or both sexes (bisex). The infection induced up-regulation of proteins associated with immune response, cytoskeletal reorganization, and apoptotic signaling. Notably, S. mansoni egg deposition led to the down-regulation of liver factors linked to energy supply and metabolic processes. Gender-specific responses were observed, with male hamsters showing higher susceptibility, supported by more differentially expressed proteins than found in females. Of note, metallothionein-2 and S100a6 proteins exhibited substantial up-regulation in livers of both genders, suggesting their pivotal roles in the liver's injury response. Immunohistochemistry and real-time-qPCR confirmed strong up-regulation of metallothionein-2 expression in the cytoplasm and nucleus upon the infection. Similar findings were seen for S100a6, which localized around granulomas and portal tracts. We also observed perturbations in metabolic pathways, including down-regulation of enzymes involved in xenobiotic biotransformation, cellular energy metabolism, and lipid modulation. Furthermore, lipidomic analyses through liquid chromatography–tandem mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry imaging identified extensive alterations, notably in cardiolipin and triacylglycerols, suggesting specific roles of lipids during pathogenesis. These findings provide unprecedented insights into the hepatic response to S. mansoni infection, shedding light on the complexity of liver pathology in this disease.

Abstract
Malignant ascites in hepatocellular carcinoma is usually a sign of advanced disease and poor prognosis and is also thought to be associated with chronic inflammation mediated by neutrophil extracellular trap (NET) networks. Although ozone, a strong oxidant, has significant antibacterial and anti-inflammatory effects, its effectiveness in treating malignant liver ascites is unclear. We first measured the levels of NETs in the peripheral blood of patients with liver cancer and healthy individuals. Next, we constructed the H22 tumor-bearing mouse model and observed the abdominal girth, body weight, survival rate, and survival time in each group; we marked the proteins associated with NETs in mouse intestinal tissues by immunofluorescence; cf-DNA and cytokines in ascites such as: tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), matrix metalloprotein 9 (MMP-9), and interferon gamma (IFN-γ) levels in ascites were measured by enzyme-linked immunosorbent assay. The expression levels of phosphorylated adenylate-activated protein kinase (P-AMPK) and scavenger receptor-A (SR-A) were detected by immunocytochemistry in the intestinal tissues of each group of mice. We further examined the expression of P-AMPK and SR-A proteins in ascites deposits by Western blotting. The results show, the plasma levels of NETs were higher in patients with hepatocellular carcinoma than in normal subjects (P < 0.01). Abdominal girth and body weight were significantly reduced in the ozone-treated group compared with the model group, while survival and survival time were dose dependently increased (both P < 0.05). NET-associated guanine histone H3 and myeloperoxidase were abundantly expressed at neutrophil aggregates in the intestinal tissues of the model mice, whereas their expression was significantly reduced in the ozone-treated group. The levels of cf-DNA, IL-6, IFN-γ, MMP-9, VEGF, and TNF-α were dose dependently increased in the ascites of H22 tumor-bearing mice in the ozone-treated group compared with the model group (all P < 0.01), while the expression of P-AMPK and SR-A proteins was increased in the ozone-treated group compared with the model group. Ozone showed significant antiperitoneal fluid production properties in H22 tumor-bearing mice, and ozone reduced peritoneal fluid production by activating AMPK and up-regulating SR-A phagocytosis damage-associated molecular patterns to reduce the production of NETs. This suggests that ozone could be used as a new drug for the treatment of malignant ascites in hepatocellular carcinoma.

Abstract
Wound healing is an intensely studied topic involved in many relevant pathophysiological processes, including fibrosis. Despite the large interest in fibrosis, the network that is related to commensal microbiota and skin fibrosis remains mysterious. Here, we pay attention to keloid, a classical yet intractable skin fibrotic disease to establish the association between commensal microbiota to scaring tissue. Our histological data reveal the presence of microbiota in the keloids. 16S rRNA sequencing characterizes microbial composition and divergence between the pathological and normal skin tissues. Moreover, the data show elevation of interleukin-8 (IL-8) in both the circulation and keloid tissue, which elicited the collagen accumulation and migratory program of dermal fibroblasts via CXCR1/2 receptor. Our research provides insights into the pathology of human fibrotic diseases, advocating commensal bacteria and IL-8 signaling as useful targets in future interventions of recurrent keloid disease.

Abstract
HIV-1 replication is durably controlled without antiretroviral therapy (ART) in certain infected individuals called elite controllers (ECs). These individuals express specific human leukocyte antigens (HLA) that tag HIV-infected cells for elimination by presenting viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In HIV-infected individuals expressing HLA-B27, CTLs primarily target the viral capsid protein (CA)-derived KK10 epitope. While selection of CA mutation R264K helps HIV-1 escape this potent CTL response, the accompanying fitness cost severely diminishes virus infectivity. Interestingly, selection of a compensatory CA mutation S173A restores HIV-1 replication. However, the molecular mechanism(s) underlying HIV-1 escape from this ART-free virus control by CTLs is not fully understood. Here, we report that the R264K mutation-associated infectivity defect arises primarily from impaired HIV-1 DNA integration, which is restored by the S173A mutation. Unexpectedly, the integration defect of the R264K variant was also restored upon depletion of the host cyclophilin A. These findings reveal a nuclear crosstalk between CA and HIV-1 integration as well as identify a previously unknown role of cyclophilin A in viral DNA integration. Finally, our study identifies a novel immune escape mechanism of an HIV-1 variant escaping a CA-directed CTL response.

Many infectious diseases, including COVID-19, are transmitted by airborne pathogens. There is a need for effective environmental control measures which, ideally, are not reliant on human behaviour. One potential solution is Krypton Chloride (KrCl) excimer lamps (often referred to as Far-UVC), which can efficiently inactivate pathogens, such as coronaviruses and influenza, in air. Research demonstrates that when KrCl lamps are filtered to remove longer-wavelength ultraviolet emissions they do not induce acute reactions in the skin or eyes, nor delayed effects such as skin cancer. While there is laboratory evidence for Far-UVC efficacy, there is limited evidence in full-sized rooms. For the first time, we show that Far-UVC deployed in a room-sized chamber effectively inactivates aerosolised Staphylococcus aureus. At a room ventilation rate of 3 air-changes-per-hour (ACH), with 5 filtered-sources the steady-state pathogen load was reduced by 98.4% providing an additional 184 equivalent air changes (eACH). This reduction was achieved using Far-UVC irradiances consistent with current American Conference of Governmental Industrial Hygienists threshold limit values for skin for a continuous 8-h exposure. Our data indicate that Far-UVC is likely to be more effective against common airborne viruses, including SARS-CoV-2, than bacteria and should thus be an effective and "hands-off" technology to reduce airborne disease transmission. The findings provide room-scale data to support the design and development of effective Far-UVC systems.

Funding: We acknowledge the financial assistance of the United Kingdom's Department for Health and Social Care (2020/092). ; Many infectious diseases, including COVID-19, are transmitted by airborne pathogens. There is a need for effective environmental control measures which, ideally, are not reliant on human behaviour. One potential solution is Krypton Chloride (KrCl) excimer lamps (often referred to as Far-UVC), which can efficiently inactivate pathogens, such as coronaviruses and influenza, in air. Research demonstrates that when KrCl lamps are filtered to remove longer-wavelength ultraviolet emissions they do not induce acute reactions in the skin or eyes, nor delayed effects such as skin cancer. While there is laboratory evidence for Far-UVC efficacy, there is limited evidence in full-sized rooms. For the first time, we show that Far-UVC deployed in a room-sized chamber effectively inactivates aerosolised Staphylococcus aureus. At a room ventilation rate of 3 air-changes-per-hour (ACH), with 5 filtered-sources the steady-state pathogen load was reduced by 98.4% providing an additional 184 equivalent air changes (eACH). This reduction was achieved using Far-UVC irradiances consistent with current American Conference of Governmental Industrial Hygienists threshold limit values for skin for a continuous 8-h exposure. Our data indicate that Far-UVC is likely to be more effective against common airborne viruses, including SARS-CoV-2, than bacteria and should thus be an effective and "hands-off" technology to reduce airborne disease transmission. The findings provide room-scale data to support the design and development of effective Far-UVC systems. ; Publisher PDF ; Peer reviewed

Abstract
Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Abstract
The primary forms of cicatricial (scarring) alopecia (PCA) are a group of inflammatory, irreversible hair loss disorders characterized by immune cell infiltrates targeting hair follicles (HFs). Lichen planopilaris (LPP), frontal fibrosing alopecia (FFA), and centrifugal cicatricial alopecia (CCCA) are among the main subtypes of PCAs. The pathogenesis of the different types of PCAs are poorly understood, and current treatment regimens yield inconsistent and unsatisfactory results. We performed high-throughput RNA-sequencing on scalp biopsies of a large cohort PCA patients to develop gene expression-based signatures, trained into machine-learning-based predictive models and pathways associated with dysregulated gene expression. We performed morphological and cytokine analysis to define the immune cell populations found in PCA subtypes. We identified a common PCA gene signature that was shared between LPP, FFA, and CCCA, which revealed a significant over-representation of mast cell (MC) genes, as well as downregulation of cholesterogenic pathways and upregulation of fibrosis and immune signaling genes. Immunohistological analyses revealed an increased presence of MCs in PCAs lesions. Our gene expression analyses revealed common pathways associated with PCAs, with a strong association with MCs. The indistinguishable differences in gene expression profiles and immune cell signatures between LPP, FFA, and CCCA suggest that similar treatment regimens may be effective in treating these irreversible forms of hair loss.

Abstract
Glioblastoma multiforme (GBM) is a highly lethal human cancer thought to originate from a self-renewing and therapeutically-resistant population of glioblastoma stem cells (GSCs). The intrinsic mechanisms enacted by GSCs during 3D tumor formation, however, remain unclear, especially in the stages prior to angiogenic/immunological infiltration. In this study, we performed a deep characterization of the genetic, immune, and metabolic profiles of GBM organoids from several patient-derived GSCs (GBMO). Despite being devoid of immune cells, transcriptomic analysis across GBMO revealed a surprising immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We find two important cell populations thought to drive GBM progression, Special AT-rich sequence-binding protein 2 (SATB2+) and homeodomain-only protein homeobox (HOPX+) progenitors, contribute to this immune landscape in GBMO and GBM in vivo. These progenitors, but not other cell types in GBMO, are resistant to conventional GBM therapies, temozolomide and irradiation. Our work defines a novel intrinsic immune-like landscape in GBMO driven, in part, by SATB2+ and HOPX+ progenitors and deepens our understanding of the intrinsic mechanisms utilized by GSCs in early GBM formation.

Abstract
The type IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbors a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX's effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX's dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the nonpathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is associated with a vast stromal reaction that arises mainly from cancer-associated fibroblasts (CAFs) and promotes both immune escape and tumor growth. Here, we used a mouse model with deletion of the activin A receptor ALK4 in the context of the KrasG12D mutation, which strongly drives collagen deposition that leads to tissue stiffness. By ligand–receptor analysis of single-cell RNA-sequencing data, we identified that, in stiff conditions, neoplastic ductal cells instructed CAFs through sustained platelet-derived growth factor (PDGF) signaling. Tumor-associated tissue rigidity resulted in the emergence of stiffness-induced CAFs (siCAFs) in vitro and in vivo. Similar results were confirmed in human data. siCAFs were able to strongly inhibit CD8+ T-cell responses in vitro and in vivo, promoting local immunosuppression. More importantly, targeting PDGF signaling led to diminished siCAF and reduced tumor growth. Our data show for the first time that early paracrine signaling leads to profound changes in tissue mechanics, impacting immune responses and tumor progression. Our study highlights that PDGF ligand neutralization can normalize the tissue architecture independent of the genetic background, indicating that finely tuned stromal therapy may open new therapeutic avenues in pancreatic cancer.

Abstract
Dental caries is a microbial disease and the most common chronic health condition, affecting nearly 3.5 billion people worldwide. In this study, we used a multiomics approach to characterize the supragingival plaque microbiome of 91 Australian children, generating 658 bacterial and 189 viral metagenome-assembled genomes with transcriptional profiling and gene-expression network analysis. We developed a reproducible pipeline for clustering sample-specific genomes to integrate metagenomics and metatranscriptomics analyses regardless of biosample overlap. We introduce novel feature engineering and compositionally-aware ensemble network frameworks while demonstrating their utility for investigating regime shifts associated with caries dysbiosis. These methods can be applied when differential abundance modeling does not capture statistical enrichments or the results from such analysis are not adequate for providing deeper insight into disease. We identified which organisms and metabolic pathways were central in a coexpression network as well as how these networks were rewired between caries and caries-free phenotypes. Our findings provide evidence of a core bacterial microbiome that was transcriptionally active in the supragingival plaque of all participants regardless of phenotype, but also show highly diagnostic changes in the ways that organisms interact. Specifically, many organisms exhibit high connectedness with central carbon metabolism to Cardiobacterium and this shift serves a bridge between phenotypes. Our evidence supports the hypothesis that caries is a multifactorial ecological disease.

Abstract
SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

Abstract
Neutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests (VNTs) currently in use are mostly surrogate tests based on direct or competitive enzyme immunoassays or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome, and thus are noninfectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) showed excellent specificity and sensitivity and correlated very well with a cVNT using fully infectious SARS-CoV-2. The results also demonstrated the reduced neutralizing capacity of COVID-19 vaccinee sera against variants of concern of SARS-CoV-2 including omicron B.1.1.529, BA.1.

Abstract
The matrix metalloproteinase MMP14 is a ubiquitously expressed, membrane-bound, secreted endopeptidase that proteolyzes substrates to regulate development, signaling, and metabolism. However, the spatial and contextual events inciting MMP14 activation and its metabolic sequelae are not fully understood. Here, we introduce an inducible, hepatocyte-specific MMP14-deficient model (MMP14LKO mice) to elucidate cell-intrinsic and systemic MMP14 function. We show that hepatocyte MMP14 mediates diet-induced body weight gain, peripheral adiposity, and impaired glucose homeostasis and drives diet-induced liver triglyceride accumulation and induction of hepatic inflammatory and fibrotic gene expression. Single-nucleus RNA sequencing revealed that hepatocyte MMP14 mediates Kupffer cell and T-cell accumulation and promotes diet-induced hepatocellular subpopulation shifts toward protection against lipid absorption. MMP14 co-immunoprecipitation and proteomic analyses revealed MMP14 substrate binding across both inflammatory and cytokine signaling, as well as metabolic pathways. Strikingly, hepatocyte MMP14 loss-of-function suppressed skeletal muscle and adipose inflammation in vivo, and in a reductionist adipose–hepatocyte co-culture model. Finally, we reveal that trehalose-type glucose transporter inhibitors decrease hepatocyte MMP14 gene expression and nominate these inhibitors as translatable therapeutic metabolic agents. We conclude that hepatocyte MMP14 drives liver and inter-organ inflammatory and metabolic sequelae of obesogenic dietary insult. Modulating MMP14 activation and blockade thus represents a targetable node in the pathogenesis of hepatic inflammation.