Deregulated immune cell recruitment orchestrated by c-MET impairs pulmonary inflammation and fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) represents the pathologic end stage of several interstitial lung diseases (ILDs) associated with high morbidity and mortality rates. However, current treatments can only delay disease progression rather than provide a cure. The role of inflammation in PF progression is well-established, but new insights into immune regulation are fundamental for developing more efficient therapies. c-MET signaling has been implicated in the migratory capacity and effector functions of immune cells. Nevertheless, the role of this signaling pathway in the context of PF-associated lung diseases remains unexplored. METHODS: To determine the influence of c-MET in immune cells in the progression of pulmonary fibrosis, we used a conditional deletion of c-Met in immune cells. To induce pulmonary fibrosis mice were administered with bleomycin (BLM) intratracheally. Over the course of 21 days, mice were assessed for weight change, and after euthanasia at different timepoints, bronchoalveolar lavage fluid cells and lung tissue were assessed for inflammation and fibrosis. Furthermore, c-MET expression was assessed in cryobiopsy sections, bronchoalveolar lavage fluid cells samples and single cell RNA-sequencing dataset from human patients with distinct interstitial lung diseases. RESULTS: c-MET expression was induced in lung immune cells, specifically in T cells, interstitial macrophages, and neutrophils, during the inflammatory phase of BLM-induced PF mouse model. Deletion of c-Met in immune cells correlated with earlier weight recovery and improved survival of BLM-treated mice. Moreover, the deletion of c-Met in immune cells was associated with early recruitment of the immune cell populations, normally found to express c-MET, leading to a subsequent attenuation of the cytotoxic and proinflammatory environment. Consequently, the less extensive inflammatory response, possibly coupled with tissue repair, culminated in less exacerbated fibrotic lesions. Furthermore, c-MET expression was up-regulated in lung T cells from patients with fibrosing ILD, suggesting a potential involvement of c-MET in the development of fibrosing disease. CONCLUSIONS: These results highlight the critical contribution of c-MET signaling in immune cells to their enhanced uncontrolled recruitment and activation toward a proinflammatory and profibrotic phenotype, leading to the exacerbation of lung injury and consequent development of fibrosis.

Profiling Bacteria in the Lungs of Patients with Severe Influenza Versus COVID-19 with or without Aspergillosis.

RATIONALE: The influence of the lung bacterial microbiome, including potential pathogens, in patients with influenza- or COVID-19-associated pulmonary aspergillosis (IAPA or CAPA) is yet to be explored. OBJECTIVES: To explore the composition of the lung bacterial microbiome and its association with viral and fungal infection, immunity and outcome in severe influenza versus COVID-19 with or without aspergillosis. METHODS: We performed a retrospective study in mechanically ventilated influenza and COVID-19 patients with or without invasive aspergillosis in whom bronchoalveolar lavage (BAL) for bacterial culture (with or without PCR) was obtained within two weeks after ICU admission. Additionally, 16S rRNA gene sequencing data and viral and bacterial load of BAL samples from a subset of these patients, and of patients requiring non-invasive ventilation, were analyzed. We integrated 16S rRNA gene sequencing data with existing immune parameter datasets. MEASUREMENTS AND MAIN RESULTS: Potential bacterial pathogens were detected in 20% (28/142) of influenza and 37% (104/281) of COVID-19 patients, while aspergillosis was detected in 38% (54/142) of influenza and 31% (86/281) of COVID-19 patients. A significant association between bacterial pathogens in BAL and 90-day mortality was found only in influenza patients, particularly IAPA patients. COVID-19 but not influenza patients showed increased pro-inflammatory pulmonary cytokine responses to bacterial pathogens. CONCLUSIONS: Aspergillosis is more frequently detected in lungs of severe influenza patients than bacterial pathogens. Detection of bacterial pathogens associates with worse outcome in influenza patients, particularly in those with IAPA, but not in COVID-19 patients. The immunological dynamics of tripartite viral-fungal-bacterial interactions deserve further investigation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Efficient small fragment sequencing of human, cow, and bison miRNA, small RNA or csRNA-seq libraries using AVITI.

Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNA) molecules including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or "nascent" RNA polymerase II transcription initiation in tissues and clinical samples. Here, we report a new protocol for seamlessly sequencing short DNA fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus) and the bison (Bison bison) sRNA or csRNA sequencing libraries, augmenting the confidence in both approaches. Additionally, analysis of generated nascent transcription start sites (TSSs) data for cattle and bison revealed inaccuracies in their current genome annotations and highlighted the possibility and need to translate small RNA sequencing methodologies to livestock. Our accelerated and optimized protocol therefore bridges the advantages of AVITI sequencing and critical methods that rely on sequencing short DNA fragments.

Higher Levels of Cytokines in Patients with Chronic Limb-Threatening Ischemia.

BACKGROUND: Inflammation is a key element in the initiation and progression of peripheral arterial disease (PAD). Understanding the impact of inflammatory molecules, as cytokines in PAD could help us to improve the prognosis of these patients. The main goal of this study was to compare the serum level of cytokines between patients with claudication to those with chronic limb-threatening ischemia (CLTI). The second objective was to evaluate the relationship between the levels of cytokines and death or amputation rate. METHODS: An observational, single-center, and prospective study was conducted from January 2018 to July 2022. The study was approved by the ethical commission of the Local Hospital (75/2017). Patients with PAD, suggested by the clinical history and objective examination and confirmed with ankle-brachial index, attending vascular surgery consultations of the first author were included. The following exclusion criteria were applied: i) bedridden individuals or subjects who refused to participate in the protocol; ii) diseases responsible for body composition changes or proinflammatory state; iii) recent diet change, iv) active malignancy, v) autoimmune disease, vi) active infection, vii) chronic renal failure (glomerular filtration rate <30 mL/min/1.73 m2), or viii) heart failure in the past 3 months. This cohort was observed at admission, 3, 6, and 12 months. A panel of 27 cytokines was determined with ELISA, at baseline. RESULTS: We included 119 subjects (mean age: 67.58 ± 9.60 years old; 79.80% males), 65 patients with claudication and 54 with CLTI. From the 27 cytokines analyzed, patients with CLTI, when compared to those with claudication, had a higher serum level of 11 cytokines: IL1ra, IL-6, IL-8, IL12 p70, G-CSF, IP-10, MCP-1, MIP-1α, PDGF-ß, RANTES, and TNF-α. From the group of patients with CLTI those who underwent a major amputation had a higher serum level of FGF-basic [median = 49.04; interquartile range = 37.03-52.49; versus median = 33.04; interquartile range = 28.60-38.98; P = 0.001]. CONCLUSIONS: Patients with CLTI have higher serum level of inflammatory cytokines, which may have role in the prognosis of these patients.

Humoral pathways of innate immune regulation in granuloma formation.

The humoral arm of mammalian innate immunity regulates several molecular mechanisms involved in resistance to pathogens, inflammation, and tissue repair. Recent studies highlight the crucial role played by humoral mediators in granulomatous inflammation. However the molecular mechanisms linking the function of these soluble molecules to the initiation and maintenance of granulomas remain elusive. We propose that humoral innate immunity coordinates fundamental physiological processes in macrophages which, in turn, initiate activation and transformation events that enable granuloma formation. We discuss the involvement of humoral mediators in processes such as immune activation, phagocytosis, metabolism, and tissue remodeling, and how these can dictate macrophage functionality during granuloma formation. These advances present opportunities for discovering novel disease factors and developing targeted, more effective treatments for granulomatous diseases.

Integrating genetic and immune factors to uncover pathogenetic mechanisms of viral-associated pulmonary aspergillosis.

Invasive pulmonary aspergillosis is a severe fungal infection primarily affecting immunocompromised patients. Individuals with severe viral infections have recently been identified as vulnerable to developing invasive fungal infections. Both influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) are linked to high mortality rates, emphasizing the urgent need for an improved understanding of disease pathogenesis to unveil new molecular targets with diagnostic and therapeutic potential. The recent establishment of animal models replicating the co-infection context has offered crucial insights into the mechanisms that underlie susceptibility to disease. However, the development and progression of human viral-fungal co-infections exhibit a significant degree of interindividual variability, even among patients with similar clinical conditions. This observation implies a significant role for host genetics, but information regarding the genetic basis for viral-fungal co-infections is currently limited. In this review, we discuss how genetic factors known to affect either antiviral or antifungal immunity could potentially reveal pathogenetic mechanisms that predispose to IAPA or CAPA and influence the overall disease course. These insights are anticipated to foster further research in both pre-clinical models and human patients, aiming to elucidate the complex pathophysiology of viral-associated pulmonary aspergillosis and contributing to the identification of new diagnostic and therapeutic targets to improve the management of these co-infections.

Inflammatory Cells in Adipose Tissue and Skeletal Muscle of Patients with Peripheral Arterial Disease or Chronic Venous Disease: A Prospective, Observational, and Histological Study.

The main goal of this study was to assess whether the presence of peripheral arterial disease (PAD) correlates with increased inflammatory cell infiltration. An observational, single-centre, and prospective study was conducted from January 2018 to July 2022. Clinical characteristics and anthropometric measures were registered. Consecutive PAD patients with surgical indications for a common femoral artery approach and patients with varicose veins with an indication for surgical ligation of the saphenofemoral junction were included. In both groups, samples of sartorius skeletal muscle, subcutaneous adipose tissue (SAT), and perivascular adipose tissue (PVAT) were collected from the femoral region. We analysed the characteristics of adipocytes and the presence of haemorrhage and inflammatory cells in the samples of PVAT and SAT via haematoxylin-eosin staining. We found that patients with PAD had significantly more inflammatory cells in PVAT [16 (43.24%) vs. 0 (0%) p = 0.008]. Analysing SAT histology, we observed that patients with PAD had significantly more CD45+ leucocytes upon immunohistochemical staining [32 (72.73%) vs. 3 (27.27%) p = 0.005]. Upon analysing skeletal muscle histology with haematoxylin-eosin staining, we evaluated skeletal fibre preservation, as well as the presence of trauma, haemorrhage, and inflammatory cells. We registered a significantly higher number of inflammatory cells in patients with PAD [well-preserved skeletal fibres: PAD = 26 (63.41%) vs. varicose veins = 3 (37.50%) p = 0.173; trauma: PAD = 4 (9.76%) vs. varicose veins = 2 (25.00%) p = 0.229; haemorrhage: PAD = 6 (14.63%) vs. varicose veins = 0 (0%) p = 0.248; inflammatory cells: PAD = 18 (43.90%) vs. varicose veins = 0 (0%) p = 0.018]. Patients with PAD had a higher number of inflammatory cells in skeletal muscle and adipose tissue (PVAT and SAT) when compared with those with varicose veins, emphasizing the role of inflammation in this group of patients.

Reversion of the Inflammatory Markers in Patients With Chronic Limb-Threatening Ischemia.

BACKGROUND: Peripheral artery disease is characterized by an intense inflammatory process that can be associated with a higher mortality rate, particularly in chronic limb-threatening ischemia (CLTI). This study aims to compare the evolution of inflammatory markers between patients with claudication with those with CLTI at 3, 6, and 12 months. METHODS AND RESULTS: An observational, single-center, and prospective study was conducted. A total of 119 patients with peripheral artery disease (65 with claudication and 54 with CLTI) were observed and inflammatory markers collected at admission and 3, 6, and 12 months. At admission, patients with CLTI, when compared with patients with claudication, had significantly higher serum levels of C-reactive protein and fibrinogen (positive acute-phase proteins) and lower serum level of albumin, total cholesterol, and high-density lipoprotein (negative acute-phase proteins): C-reactive protein (g/dL), 2.90 (25th-75th percentile, 2.90-4.90) versus 6.80 (25th-75th percentile, 2.90-53.26) (P=0.000); fibrinogen (mg/dL), 293.00 (25th-75th percentile, 269.25-349.00) versus 415.50 (25th-75th percentile, 312.00-615.75) (P=0.000); total cholesterol (mg/dL), 161.79±95% [152.74-170.85] versus 146.42%±95% [135.30-157.53] (P=0.034); high-density lipoprotein (mg/dL), 50.00 (25th-75th percentile, 41.00-60.00) versus 37.00 (25th-75th percentile, 30.00-45.50) (P=0.000); albumin (g/dL): 4.00 (25th-75th percentile, 3.70-4.20) versus 3.60 (25th-75th percentile, 3.10-4.00) (P=0.003). The association between CLTI and total cholesterol was lost after adjusting for confounders. Three months after the resolution of the CLTI, there was an increase in the levels of negative acute-phase proteins and a decrease in positive acute-phase proteins. These inflammatory proteins did not register an evolution in patients with claudication. The differences in the inflammatory proteins between groups disappeared at 6 months. CONCLUSIONS: CLTI has an inflammatory environment that can be partially reverted after resolution of the ischemic process, emphasizing the importance of timely intervention.

Akkermansia muciniphila and Parabacteroides distasonis synergistically protect from colitis by promoting ILC3 in the gut.

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the gastrointestinal tract. The etiology of IBD remains elusive, but the disease is suggested to arise from the interaction of environmental and genetic factors that trigger inadequate immune responses and inflammation in the intestine. The gut microbiome majorly contributes to disease as an environmental variable, and although some causative bacteria are identified, little is known about which specific members of the microbiome aid in the intestinal epithelial barrier function to protect from disease. While chemically inducing colitis in mice from two distinct animal facilities, we serendipitously found that mice in one facility showed remarkable resistance to disease development, which was associated with increased markers of epithelial barrier integrity. Importantly, we show that Akkermansia muciniphila and Parabacteroides distasonis were significantly increased in the microbiota of resistant mice. To causally connect these microbes to protection against disease, we colonized susceptible mice with the two bacterial species. Our results demonstrate that A. muciniphila and P. distasonis synergistically drive a protective effect in both acute and chronic models of colitis by boosting the frequency of type 3 innate lymphoid cells in the colon and by improving gut epithelial integrity. Altogether, our work reveals a combined effort of commensal microbes in offering protection against severe intestinal inflammation by shaping gut immunity and by enhancing intestinal epithelial barrier stability. Our study highlights the beneficial role of gut bacteria in dictating intestinal homeostasis, which is an important step toward employing microbiome-driven therapeutic approaches for IBD clinical management. IMPORTANCE: The contribution of the gut microbiome to the balance between homeostasis and inflammation is widely known. Nevertheless, the etiology of inflammatory bowel disease, which is known to be influenced by genetics, immune response, and environmental cues, remains unclear. Unlocking novel players involved in the dictation of a protective gut, namely, in the microbiota component, is therefore crucial to develop novel strategies to tackle IBD. Herein, we revealed a synergistic interaction between two commensal bacterial strains, Akkermansia muciniphila and Parabacteroides distasonis, which induce protection against both acute and chronic models of colitis induction, by enhancing epithelial barrier integrity and promoting group 3 innate lymphoid cells in the colonic mucosa. This study provides a novel insight on how commensal bacteria can beneficially act to promote intestinal homeostasis, which may open new avenues toward the use of microbiome-derived strategies to tackle IBD.

Ovine Herpesvirus 2 Glycoprotein B Complementation Restores Infectivity to a Bovine Herpesvirus 4 gB-Null Mutant.

Ovine herpesvirus 2 (OvHV-2) and bovine herpesvirus 4 (BoHV-4) are gamma herpesviruses that belong to the genera Macavirus and Rhadinovirus, respectively. As with all herpesviruses, both OvHV-2 and BoHV-4 express glycoprotein B (gB), which plays an essential role in the infection of host cells. In that context, it has been demonstrated that a BoHV-4 gB-null mutant is unable to infect host cells. In this study, we used homologous recombination to insert OvHV-2 ORF 8, encoding gB, into the BoHV-4 gB-null mutant genome, creating a chimeric BoHV-4 virus carrying and expressing OvHV-2 gB (BoHV-4∆gB/OvHV-2-gB) that was infectious and able to replicate in vitro. We then evaluated BoHV-4∆gB/OvHV-2-gB as a potential vaccine candidate for sheep-associated malignant catarrhal fever (SA-MCF), a fatal disease of ungulates caused by OvHV-2. Using rabbits as a laboratory model for MCF, we assessed the safety, immunogenicity, and efficacy of BoHV-4∆gB/OvHV-2-gB in an immunization/challenge trial. The results showed that while BoHV-4∆gB/OvHV-2-gB was safe and induced OvHV-2 gB-specific humoral immune responses, immunization conferred only 28.5% protection upon challenge with OvHV-2. Therefore, future studies should focus on alternative strategies to express OvHV-2 proteins to develop an effective vaccine against SA-MCF.

Reemerging/Notifiable Diseases to Watch.

Reemerging and notifiable diseases of cattle and bison continue to pose potential risks to their health and lives and affecting production and the livelihoods of producers. It is essential to understand the clinical presentation of these diseases to watch for possible incursions and infections and to immediately report your suspicions to your State and Federal Animal Health Officials. Three of these reemerging and notifiable diseases of cattle and bison, malignant catarrhal fever, bluetongue virus, and New World screwworm, are presented in this article for increased awareness to consider as a differential if examinations present suggestive clinical signs.

Metabolic regulation of the host-fungus interaction: from biological principles to therapeutic opportunities.

Fungal infections present a significant global public health concern, impacting over one billion individuals worldwide and resulting in more than 3 million deaths annually. Despite considerable progress in recent years, the management of fungal infections remains challenging. The limited development of novel diagnostic and therapeutic approaches is largely attributed to our incomplete understanding of the pathogenetic mechanisms involved in these diseases. Recent research has highlighted the pivotal role of cellular metabolism in regulating the interaction between fungi and their hosts. In response to fungal infection, immune cells undergo complex metabolic adjustments to meet the energy demands necessary for an effective immune response. A comprehensive understanding of the metabolic circuits governing antifungal immunity, combined with the integration of individual host traits, holds the potential to inform novel medical interventions for fungal infections. This review explores recent insights into the immunometabolic regulation of host-fungal interactions and the infection outcome and discusses how the metabolic repurposing of immune cell function could be exploited in innovative and personalized therapeutic approaches.

Coding complete genome of a strain of ovine herpesvirus-2 associated with a clinical case of malignant catarrhal fever in a domestic lamb.

Ovine herpesvirus-2 causes sheep-associated malignant catarrhal fever, a fatal disease of ruminants and pigs. The virus is carried by sheep, and infection is typically subclinical. Here, we report the coding complete genome sequence of a strain of OvHV-2 obtained from a clinically affected domestic lamb.

Enhanced Sensitivity in Optical Sensors through Self-Image Theory and Graphene Oxide Coating.

This paper presents an approach to enhancing sensitivity in optical sensors by integrating self-image theory and graphene oxide coating. The sensor is specifically engineered to quantitatively assess glucose concentrations in aqueous solutions that simulate the spectrum of glucose levels typically encountered in human saliva. Prior to sensor fabrication, the theoretical self-image points were rigorously validated using Multiphysics COMSOL 6.0 software. Subsequently, the sensor was fabricated to a length corresponding to the second self-image point (29.12 mm) and coated with an 80 µm/mL graphene oxide film using the Layer-by-Layer technique. The sensor characterization in refractive index demonstrated a wavelength sensitivity of 200 ± 6 nm/RIU. Comparative evaluations of uncoated and graphene oxide-coated sensors applied to measure glucose in solutions ranging from 25 to 200 mg/dL showed an eightfold sensitivity improvement with one bilayer of Polyethyleneimine/graphene. The final graphene oxide-based sensor exhibited a sensitivity of 10.403 ± 0.004 pm/(mg/dL) and demonstrated stability with a low standard deviation of 0.46 pm/min and a maximum theoretical resolution of 1.90 mg/dL.

Herpes Simplex Virus 1 Glycoprotein B from a Hyperfusogenic Virus Mediates Enhanced Cell-Cell Fusion.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4 °C and also entered cells more efficiently at 15 °C, relative to wild type HSV-1 strain KOS virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type KOS. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type KOS gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the KOS gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

Impact of polyurethane versus acellular dermal matrix coating on prepectoral reconstruction outcomes: Interface does matter.

BACKGROUND: Interfaces continue to be used in prepectoral breast reconstruction to refine breast appearance, but more clinical data are required to assess their effectiveness. This study compares the rates of capsular contracture, breast esthetics, and patient satisfaction between two commonly used interface materials, acellular dermal matrix (ADM) and polyurethane (PU) foam. METHODS: A cross-sectional assessment was conducted on all patients who underwent prepectoral direct-to-implant reconstruction with an interface material between June 2018 and June 2022. We compared capsular contracture rates (assessed in-person), esthetic outcomes (evaluated by a three-member panel using a specially designed scale), and patient satisfaction (measured using the Breast-Q questionnaire) among the members of the interface groups. RESULTS: Among the 79 reconstructed breasts (20 bilateral cases), 35 were reconstructed using ADM and 44 using PU implants. The ADM group had a significantly higher frequency of Baker III/IV capsular contracture compared with the PU group (14.3% vs. 0%, p = 0.014) and lower ratings from the panel in terms of capsular contracture (median 3.7 vs. 4.0, p < 0.001). PU reconstructions scored worse in implant visibility (median 2.3 vs. 3.3, p < 0.001) and rippling (median 3.0 vs. 3.7, p < 0.001). However, after appropriate adjustment for confounders, no significant differences in overall appearance and patient satisfaction were found. CONCLUSIONS: ADM reconstructions are prone to capsular contracture with all their related esthetic issues, but PU implants have certain cosmetic flaws, such as implant visibility and malposition. Since each technique has its own limitations, neither the experienced surgeons nor patients exhibited a clear preference for either approach.

Multicentric Study on the Clinical Mycology Capacity and Access to Antifungal Treatment in Portugal.

The success of the clinical management of invasive fungal diseases (IFD) is highly dependent on suitable tools for timely and accurate diagnosis for effective treatment. An in-depth analysis of the ability of European institutions to promptly and accurately diagnose IFD was previously conducted to identify limitations and aspects to improve. Here, we evaluated and discussed the specific case of Portugal, for which, to our knowledge, there are no reports describing the national mycological diagnostic capacity and access to antifungal treatment. Data from 16 Portuguese medical institutions were collected via an online electronic case report form covering different parameters, including institution profile, self-perceived IFD incidence, target patients, diagnostic methods and reagents, and available antifungals. The majority of participating institutions (69%) reported a low-very low incidence of IFD, with Candida spp. indicated as the most relevant fungal pathogen, followed by Aspergillus spp. and Cryptococcus spp. All institutions had access to culture and microscopy, whereas 94 and 88% were able to run antigen-detection assays and molecular tests, respectively. All of the institutions capable of providing antifungal therapy declared to have access to at least one antifungal. However, echinocandins were only available at 85% of the sites. Therapeutic drug monitoring (TDM) was reported to remain a very restricted practice in Portugal, being available in 19% of the institutions, with the TDM of itraconazole and posaconazole performed in only 6% of them. Importantly, several of these resources are outsourced to external entities. Except for TDM, Portugal appears to be well-prepared concerning the overall capacity to diagnose and treat IFD. Future efforts should focus on promoting the widespread availability of TDM and improved access to multiple classes of antifungals, to further improve patient outcomes.

Lower respiratory tract single-cell RNA sequencing and neutrophil extracellular trap profiling of COVID-19-associated pulmonary aspergillosis: a single centre, retrospective, observational study.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA. METHODS: We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, we determined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec 31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality. FINDINGS: We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30 male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages and monocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA. INTERPRETATION: Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19. FUNDING: Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020.

Inflammation Is a Histological Characteristic of Skeletal Muscle in Chronic Limb Threatening Ischemia.

BACKGROUND: The loss of skeletal muscle is a prognostic factor in several diseases including in patients with chronic limb threatening ischemia (CLTI). Patients with CLTI also have a lower skeletal mass and area when compared to those with claudication. However, there are no currently available data regarding the histological characteristics of core muscles in patients with CLTI. This study aims to determine the differences in core skeletal muscles between patients with claudication and those with CLTI. The second aim is to evaluate the differences in myokines, which are molecules secreted by skeletal muscle, between patients with claudication and those with CLTI. METHODS: An observational, prospective study was conducted from January 2018 to July 2022 involving consecutive patients with peripheral arterial disease (PAD). The clinical characteristics were registered. In PAD patients with surgical indication for common femoral artery approach, samples of sartorius skeletal muscle (and not from the limb muscles directly involved in the ischemic process) were collected. The samples were submitted to histological characterization on hematoxylin-eosin and to immunohistochemical analysis to detect CD45+ leukocytes and CD163+ macrophages. The extent of the inflammatory cells (leukocytes and macrophages) was semiquantitatively assessed using a 0-to-4 grade scale as follows: absent (0†), mild (†), moderate (††), severe (†††), and very severe (††††). Serum levels of myokines: irisin, myostatin, IL-8, and lL-6 were determined with multiplex bead-based immunoassay. RESULTS: 119 patients (mean age: 67.58 ± 9.60 years old, 79.80% males) 64 with claudication and 54 with CLTI were enrolled in the study. No differences were registered between patients with claudication and those with CLTI on age, gender, cardiovascular risk factors, and medication, except on smoking habits. There was a significantly higher prevalence of smokers and a higher smoking load in the claudication group. Samples of sartorius skeletal muscle from 40 patients (14 with claudication and 26 with CLTI) were submitted to histological analysis. No differences were found in skeletal muscle fibers preservation, trauma, or hemorrhage (on hematoxylin-eosin staining). However, in the immunohistochemistry study, we found more inflammatory cells CD45+ leukocytes in patients with CLTI when compared to those with claudication [CD45+ ≥ moderate (††): claudication (n = 14): 4; 28.57%; CLTI (n = 25): 16; 64.00%; P = 0.034]. Patients with CLTI also had higher tissue levels of CD163+ macrophages, but this difference was not significant [CD163+ ≥ moderate (††): claudication (n = 13): 7; 53.85%; CLTI (n = 27): 21; 77.78%; P = 0.122]. The serum levels of the myokines, irisin, and myostatin were below the lower limit of detection, in the majority of patients, so no valid results were obtained. However, patients with CLTI had a higher serum level of Interleukin (IL)-6 and IL-8. CONCLUSIONS: CLTI patients exhibit increased quantities of leukocytes in their sartorius muscle, as well as elevated serum levels of myokines IL-8 and IL-6. Inflamed skeletal muscle can contribute to the loss of muscle mass and account for the lower density of skeletal muscle observed in CLTI. Additionally, inflamed skeletal muscle may contribute to the development of systemic inflammation through the secretion of pro-inflammatory cytokines into the systemic circulation. Halting the inflammatory process could eventually improve the prognosis of CLTI patients.

Membrane fusion activity of herpes simplex virus 1 glycoproteins from a hyperfusogenic virus.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4°C and also entered cells more efficiently at 15°C relative to wild type virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the wild type gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. Wild type gC is proposed to facilitate fusion and entry into epithelial cells by optimizing conformational changes in the fusion protein gB. ANG gC substitution or addition also had no effect on cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.