Detection of volatile organic compounds as new paradigm to accelerate antimicrobial susceptibility testing: performance evaluation of VITEK® REVEAL™.

OBJECTIVES: The measurement of VOCs release in the headspace of a bacterial culture represents a new approach to rapidly assess antimicrobial susceptibility. Herein, we evaluated the diagnostic performance of the VITEK® REVEAL™ system directly from a collection of Gram-negative positive blood cultures. MATERIALS AND METHODS: One hundred and twenty-eight positive blood cultures were included in the analysis (Enterobacterales, n = 95; Pseudomonas aeruginosa, n = 21; Acinetobacter baumannii complex, n = 12). Samples were processed using VITEK® REVEAL™ according to the manufacturer's recommendations, and MICs of 22 antimicrobials were compared with those obtained using reference methods. Categorical agreement (CA), essential agreement (EA) and categorical errors were calculated. RESULTS: Overall, 2220 strain/antibiotic pair combinations were analysed. Of these, most were classified as resistant by reference antimicrobial susceptibility testing (1091/2220; 48.7%). The overall CA and EA were 97.6% and 97.7%, respectively. CA ranged from 97.5% in Enterobacterales to 97.9% in both P. aeruginosa and A. baumannii complex. The overall number of categorical discrepancies were: 18 very major errors (1.6%), 13 major errors (1.2%) and 22 minor errors (2.4%). EA ranged from 95.2% in P. aeruginosa to 98.1% in Enterobacterales. Screening test for ESBL phenotype was positive, indeterminate and negative in 13.7%, 32.6% and 27.4% of Enterobacterales isolates tested by both VITEK® REVEAL™ and the reference method, showing 100% CA. CONCLUSIONS: VITEK® REVEAL™ represents a reliable tool to obtain antimicrobial susceptibility results of the main Gram-negative species directly from positive blood cultures with time to results of less than 8 h.

Antimicrobial Efficacy of a Vegetable Oil Plasticizer in PVC Matrices.

The growing prevalence of bacterial and viral infections, highlighted by the recent COVID-19 pandemic, urgently calls for new antimicrobial strategies. To this end, we have synthesized and characterized a novel fatty acid epoxy-ester plasticizer for polymers, named GDE. GDE is not only sustainable and user-friendly but also demonstrates superior plasticizing properties, while its epoxy components improve the heat stability of PVC-based matrices. A key feature of GDE is its ability to confer antimicrobial properties to surfaces. Indeed, upon contact, this material can effectively kill enveloped viruses, such as herpes simplex virus type 1 (HSV-1) and the ß-coronavirus prototype HCoV-OC43, but it is ineffective against nonenveloped viruses like human adenovirus (HAdV). Further analysis using transmission electron microscopy (TEM) on HSV-1 virions exposed to GDE showed significant structural damage, indicating that GDE can interfere with the viral envelope, potentially causing leakage. Moreover, GDE demonstrates antibacterial activity, albeit to a lesser extent, against notorious pathogens such as Staphylococcus aureus and Escherichia coli. Overall, this newly developed plasticizer shows significant potential as an antimicrobial agent suitable for use in both community and healthcare settings to curb the spread of infections caused by microorganisms contaminating physical surfaces.

Extracellular microRNAs induce dendritic cell-dependent joint inflammation and potentiate osteoclast differentiation via TLR7/8 engagement.

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

Dalbavancin Boosts the Ability of Neutrophils to Fight Methicillin-Resistant Staphylococcus aureus.

Polymorphonuclear leukocytes (PMNs) are the most important cell type involved in the early nonspecific host response to bacterial pathogens. Staphylococcus aureus has evolved mechanisms to evade immune responses that contribute to its persistence in PMNs, and acquired resistance to several antimicrobials. Additionally, methicillin-resistant S. aureus (MRSA) is one of the most common causes of acute bacterial skin and skin-structure infections (ABSSSIs). Dalbavancin (DBV), a lipoglycopeptide, is indicated for the treatment of ABSSSIs, and has a broad spectrum of action against most microorganisms. Here, we sought to determine the effect of DBV on the neutrophil killing of MRSA and its potential immunomodulating activity. Our results revealed that DBV boosts MRSA killing by acting on both bacteria and PMNs. DBV pre-treatment of PMNs did not change the respiratory burst or degranulation, while an increased trend in neutrophil extracellular traps-associated elastase and in the production of TNFα and CXCL8 was revealed. In parallel, DBV caused a delay in the apoptosis of MRSA-infected neutrophils. In conclusion, we demonstrated a cooperative effect between the antimicrobial properties of PMNs and DBV, thus owing to their immunomodulatory activity. In the choice of the treatment management of serious S. aureus infections, DBV should be considered as an outstanding option since it reinforces PMNs pathogen clearance capability by exerting its effect directly, not only on MRSA but also on neutrophils.

Understanding water transport through graphene-based nanochannels via experimental control of slip length.

The water transport along graphene-based nanochannels has gained significant interest. However, experimental access to the influence of defects and impurities on transport poses a critical knowledge gap. Here, we investigate the water transport of cation intercalated graphene oxide membranes. The cations act as water-attracting impurities on the channel walls. Via water transport experiments, we show that the slip length of the nanochannels decay exponentially with the hydrated diameter of the intercalated cations, confirming that water transport is governed by the interaction between water molecules and the impurities on the channel wall. The exponential decay of slip length approximates non-slip conditions. This offers experimental support for the use of the Hagen-Poiseuille equation in graphene-based nanochannels, which was previously only confirmed by simulations. Our study gives valuable feedback to theoretical predictions of the water transport along graphene-based channels with water-attracting impurities.

Mass Transport via In-Plane Nanopores in Graphene Oxide Membranes.

Angstrom-confined solvents in 2D laminates can travel through interlayer spacings, through gaps between adjacent sheets, and via in-plane pores. Among these, experimental access to investigate the mass transport through in-plane pores is lacking. Our experiments allow an understanding of this mass transport via the controlled variation of oxygen functionalities, size and density of in-plane pores in graphene oxide membranes. Contrary to expectations, our transport experiments show that higher in-plane pore densities may not necessarily lead to higher water permeability. We observed that membranes with a high in-plane pore density but a low amount of oxygen functionalities exhibit a complete blockage of water. However, when water-ethanol mixtures with a weaker hydrogen network are used, these membranes show an enhanced permeation. Our combined experimental and computational results suggest that the transport mechanism is governed by the attraction of the solvents toward the pores with functional groups and hindered by the strong hydrogen network of water formed under angstrom confinement.

Norepinephrine and Serotonin Can Modulate the Behavior of the Probiotic Enterococcus faecium NCIMB10415 towards the Host: Is a Putative Surface Sensor Involved?

The human gut microbiota has co-evolved with humans by exchanging bidirectional signals. This study aims at deepening the knowledge of this crucial relationship by analyzing phenotypic and interactive responses of the probiotic Enterococcus faecium NCIMB10415 (E. faecium SF68) to the top-down signals norepinephrine (NE) and serotonin (5HT), two neuroactive molecules abundant in the gut. We treated E. faecium NCIMB10415 with 100 µM NE and 50 µM 5HT and tested its ability to form static biofilm (Confocal Laser Scanning Microscopy), adhere to the Caco-2/TC7 monolayer, affect the epithelial barrier function (Transepithelial Electrical Resistance) and human dendritic cells (DC) maturation, differentiation, and cytokines production. Finally, we evaluated the presence of a putative hormone sensor through in silico (whole genome sequence and protein modelling) and in vitro (Micro-Scale Thermophoresis) analyses. The hormone treatments increase biofilm formation and adhesion on Caco-2/TC7, as well as the epithelial barrier function. No differences concerning DC differentiation and maturation between stimulated and control bacteria were detected, while an enhanced TNF-α production was observed in NE-treated bacteria. Investigations on the sensor support the hypothesis that a two-component system on the bacterial surface can sense 5HT and NE. Overall, the data demonstrate that E. faecium NCIMB10415 can sense both NE and 5HT and respond accordingly.

Bartonella henselae Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cell Activation and Infectibility That Amplifies the Angiogenic Process.

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation.

Bisphenol A and S in the Urine of Newborns: Plastic for Non-Food Use Still without Rules.

The aim of the present study was to assess the effects of bisphenol (BP) exposure on pregnancy and neonatal life. We have (a) determined BP (BPA and BPS) concentration levels in a group of newborns and their mothers; (b) identified factors, habits, and devices possibly responsible for BP uptake; and (c) determined the effect of BP exposure. No significant correlations were detected between maternal and neonatal BP concentration levels. In newborns, positive correlations between pacifier use and BPS total (p = 0.04) and free BPS (p = 0.03) concentrations were detected. A significant correlation was also found between oral glucose administration and concentration levels of free BPA (p < 0.05). Our study points to a central role of lifestyle, hospital procedures, and neonatal devices in inducing BP exposure, especially during the perinatal period. This is the first report of BP contamination in newborns due to widely non-alimentary products designed for newborn care, such as glucose-solution containers for BPA and pacifiers for BPS. Further studies are advocated in order to clarify both the impact of other BP forms on human health and development, as well as potential BPA exposure sources during neonatal and childhood life.

Enhanced Antimicrobial and Antibiofilm Effect of New Colistin-Loaded Human Albumin Nanoparticles.

Multidrug-resistant (MDR) Gram-negative bacteria (GNB), such as Acinetobacter and Klebsiella, are responsible for severe hospital-acquired infections. Colistin, despite its toxicity and low tissue penetration, is considered the last resort antibiotic against these microorganisms. Of concern, the use of Colistin has recently been compromised by the emergence of Colistin resistance. Herein, we developed a new formulation consisting of multifunctional chitosan-coated human albumin nanoparticles for the delivery of Colistin (Col/haNPs). Col/haNPs were in vitro characterized for encapsulation efficiency, drug release, stability and cytotoxicity and were evaluated for antibacterial activity against MDR GNB (Acinetobacter baumannii and Klebsiella pneumoniae). Col/haNPs showed sizes lower than 200 nm, high encapsulation efficiency (98.65%) and prolonged in vitro release of Colistin. The safety of the nanoformulation was demonstrated by a negligible cytotoxicity on human fibroblasts and hemolytic activity. Col/haNPs evidenced a high antibacterial effect with a significant decrease in MIC values compared to free Colistin, in particular against Col-resistant strains with a pronounced decline of bacterial growth over time. Moreover, Col/haNPs exhibited an inhibitory effect on biofilm formation that was 4 and 60 fold higher compared to free Colistin, respectively for Colistin susceptible and resistant A. baumannii. Our findings suggest that Col/haNPs could represent a promising Colistin nanocarrier with high antimicrobial activity on MDR GNB.

Inhibition of Human Neutrophil Functions In Vitro by Multiple Sclerosis Disease-Modifying Therapies.

There is a growing optimism about the potential of new disease-modifying therapies (DMTs) in the management of relapsing-remitting multiple sclerosis (RRMS) patients. However, this initial enthusiasm has been tempered by evidence indicating that multiple sclerosis (MS) patients undergoing DMT may be at higher risk of developing infections through incompletely understood mechanisms. As neutrophils provide the first line of defense against pathogens, here we have compared the effects of some of the commonly used MS DMTs (i.e., moderate-efficacy injective, first-line: interferonß-1b (IFNß-1b), glatiramer acetate (GA); and high-efficacy, second-line: fingolimod (FTY) and natalizumab (NAT)) on the in vitro viability and functions of neutrophils isolated from healthy subjects. All the DMTs tested impaired the ability of neutrophils to kill Klebsiella pneumoniae, whereas none of them affected the rate of neutrophil apoptosis or CD11b and CD62L cell surface expression. Intriguingly, only FTY exposure negatively affected K. pneumoniae-induced production of reactive oxygen species (ROS) in polymorphonuclear leukocytes (PMNs). Furthermore, neutrophils exposed to K. pneumoniae secreted enhanced amounts of CXCL8, IL-1ß and TNF-α, which were differentially regulated following DMT pretreatment. Altogether, these findings suggest that DMTs may increase the susceptibility of MS patients to microbial infections, in part, through inhibition of neutrophil functions. In light of these data, we recommend that the design of personalized therapies for RRMS patients should take into account not just the mechanism of action of the chosen DMT but also the potential risk of infection associated with the administration of such therapeutic compounds to this highly vulnerable population.

Selective Proton Transport for Hydrogen Production Using Graphene Oxide Membranes.

Graphene oxide has shown exceptional properties in terms of water permeability and filtration characteristics. Here the suitability of graphene oxide membranes for the spatial separation of hydronium and hydroxide ions after photocatalytic water splitting is demonstrated. Instead of relying on classical size exclusion by adjusting the membrane laminates' interlayer spacings, nonmodified graphene oxide is used to exploit the presence of its natural functional groups and surface charges for filtration. Despite a significantly larger interlayer spacing inside the membrane compared with the size of the hydrated radii of the ions, highly asymmetric transport behavior and a 6 times higher mobility for hydronium than for hydroxide are observed. DFT simulations reveal that hydroxide ions are more prone to interact and stick to the functional groups of graphene oxide, while diffusion of hydronium ions through the membrane is less impeded and aligns well with the concept of the Grotthuss mechanism.

CSF1R Is Required for Differentiation and Migration of Langerhans Cells and Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disorder characterized by tissue accumulation of CD1a+CD207+ LCH cells. In LCH, somatic mutations of the BRAF V600E gene have been detected in tissue LCH cells, bone marrow CD34+ hematopoietic stem cells, circulating CD14+ monocytes, and BDCA1+ myeloid dendritic cells (DC). Targeting BRAF V600E in clonal Langerhans cells (LC) and their precursors is a potential treatment option for patients whose tumors have the mutation. The development of mouse macrophages and LCs is regulated by the CSF1 receptor (CSF1R). In patients with diffuse-type tenosynovial giant cell tumors, CSF1R inhibition depletes tumor-associated macrophages (TAM) with therapeutic efficacy; however, CSF1R signaling in LCs and LCH has not been investigated. We found through IHC and flow cytometry that CSF1R is normally expressed on human CD1a+CD207+ LCs in the epidermis and stratified epithelia. LCs that were differentiated from CD14+ monocytes, BDCA1+ DCs, and CD34+ cord blood progenitors expressed CSF1R that was downregulated upon maturation. Immature LCs migrated toward CSF1, but not IL34. Administration of the c-FMS/CSF1R kinase inhibitors GW2580 and BLZ945 significantly reduced human LC migration. In LCH clinical samples, LCH cells (including BRAF V600E cells) and TAMs retained high expression of CSF1R. We also detected the presence of transcripts for its ligand, CSF1, but not IL34, in all tested LCH cases. CSF1R and CSF1 expression in LCH, and their role in LC migration and differentiation, suggests CSF1R signaling blockade as a candidate rational approach for treatment of LCH, including the BRAF V600E and wild-type forms of the disease.

Impact of imipenem concentration in lung perfusate and tissue biopsy during clinical ex-vivo lung perfusion of high-risk lung donors.

BACKGROUND: Normothermic ex-vivo lung perfusion (EVLP) limits organ donor shortage by potentially using high-risk donor lungs. Microbial burden reduction has been demonstrated after EVLP using antibiotic prophylaxis with imipenem. However, no data have been published on the clinical consequences of the potential residual bacterial burden. METHODS: Imipenem concentration was measured every hour (T0 to T6) in the lung perfusate and at the end of EVLP (Tf) in biopsies. The antimicrobial activity of perfusate at T1 and Tf against E. coli and K. pneumoniae was evaluated. Lungs were distinguished: no bacterial species in recipients and donors (donor-/recipient-); bacterial species isolated from donors and not from recipients (donor+/recipient-); same bacterial species in both recipients and donors (donor+/recipient+). Interleukin 6 (IL-6) and IL-8 concentrations in lung perfusate, clinical pulmonary infection score (CPIS) and primary graft dysfunction (PGD) were evaluated. RESULTS: Imipenem concentration in perfusate decreased over time. T1 and Tf perfusates exhibited bactericidal activity against E. coli and K. pneumoniae. Overall, T1 perfusates yielded higher bactericidal titers (BTs) than Tf. The donor+/recipient+ group (26% of cases) had higher IL-6 and IL-8 in perfusate and higher CPIS. CONCLUSIONS: Recipients with the same bacterial species isolated in their donors had higher risk of pulmonary inflammation and early post-transplant pneumonia. Improvements in antimicrobial strategies during EVLP are warranted to minimize the consequences of donor associated respiratory infection.

Citrate anion improves chronic dialysis efficacy, reduces systemic inflammation and prevents Chemerin-mediated microvascular injury.

Systemic inflammation and uremic toxins (UT) determine the increased cardiovascular mortality observed in chronic hemodialysis (HD) patients. Among UT, the adipokine Chemerin induces vascular dysfunction by targeting both endothelial and vascular smooth muscular cells (EC and VSMC). As Citrate anion modulates oxidative metabolism, systemic inflammation and vascular function, we evaluated whether citrate-buffered dialysis improves HD efficiency, inflammatory parameters and chemerin-mediated microvascular injury. 45 patients were treated in sequence with acetate, citrate and, again, acetate-buffered dialysis solution (3 months per interval). At study admission and after each treatment switch, we evaluated dialysis efficacy and circulating levels of chemerin and different inflammatory biomarkers. In vitro, we stimulated EC and VSMC with patients' plasma and we investigated the role of chemerin as UT. Citrate dialysis increased HD efficacy and reduced plasma levels of CRP, fibrinogen, IL6 and chemerin. In vitro, patients' plasma induced EC and VSMC dysfunction. These effects were reduced by citrate-buffered solutions and paralleled by the decrease of chemerin levels. Consistently, chemerin receptor knockdown reduced EC and VSMC dysfunction. In conclusion, Switching from acetate to citrate improved dialysis efficacy and inflammatory parameters; in vitro, chemerin-induced EC and VSMC injury were decreased by using citrate as dialysis buffer.

Role of osteopontin in dendritic cell shaping of immune responses.

Osteopontin (OPN) is a pleiotropic cytokine produced both by immune and non-immune cells and active on different cellular targets. OPN production has been associated with several pathological conditions, including autoimmune diseases (e.g. lupus, multiple sclerosis and rheumatoid arthritis) and cancer. Emerging evidence suggests that the role of OPN has been underestimated, as it seems to be working at multiple levels of immune regulation, such as the shaping of T cell effector responses, the regulation of the tumor microenvironment, and the functional interaction with mesenchymal stromal cells. In this context, dendritic cells (DCs) play a crucial role being both an important source and a cellular target for OPN action. DC family is composed by several cell subsets endowed with specific immune functions. OPN exerts its biological functions through multiple receptors and is produced in different intracellular and secreted forms. OPN production by DC subsets is emerging as a crucial mechanism of regulation in normal and pathological conditions and starts to be exploited as a therapeutic target. This review will focus on the role of DC-derived OPN in shaping immune response and on the complex role of this cytokines in the regulation in immune response.

Atomistic Simulations in Surface Chemistry to Interpret Scanning Probe Microscopy Images: A Short Review.

Atomistic simulations are a powerful tool to explain and guide experimental investigations, but there are cases where a clear correspondence is difficult to obtain. While the theoretical framework to get the static picture of the equilibrium structures in vacuum is well-established, it is challenging to correctly model them in operando conditions (at the right experimental temperature, pH and pressure). In this short review the main theoretical approaches are briefly presented, supported by selected case studies where the structural and dynamical properties of different systems are investigated. A successful match with the experimental data is accomplished by choosing the proper level of theory in order to describe the structure under study in the most accurate and realistic way.

In Vitro Enhanced Skin Permeation and Retention of Imiquimod Loaded in ß-Cyclodextrin Nanosponge Hydrogel.

Imiquimod (IMQ) is an immune response modifier clinically used for the treatment of various topical diseases. However, its poor aqueous solubility and skin penetration capability make the topical delivery of IMQ a challenging task. This work aims at developing a nanomedicine-based topical formulation, carrying IMQ to control the scarring process for the treatment of aberrant wounds. For this purpose, IMQ was loaded in ß-cyclodextrin-based nanosponges and dispersed in a hydrogel suitable for dermal application. The formulation was characterized in vitro and compared with IMQ inclusion complexes, with (2-hydroxy)propyl ß-cyclodextrin(HPßCD) and carboxymethyl ß-cyclodextrin (CMßCD) showing enhanced penetration properties. The hydrogel containing IMQ-loaded nanosponges could act as a drug reservoir and guarantee the sustained release of IMQ through the skin. A greater inhibitory effect on fibroblast proliferation was observed for IMQ loaded in nanosponges compared to the other formulations.

Serological and molecular detection of Bartonella henselae in specimens from patients with suspected cat scratch disease in Italy: A comparative study.

Cat scratch disease (CSD) is an infectious disease caused by Bartonella henselae, usually characterized by self-limiting regional lymphadenopathy and fever. Given the low clinical diagnostic sensitivity and specificity of conventional anti-B. henselae indirect immunofluorescence assays (IFAs), real-time polymerase chain reaction (PCR)-based detection of B. henselae is now being proposed as a more sensitive tool to diagnose CSD. Thus, here we have assessed the efficacy of real-time PCR in detecting B. henselae in different specimens from patients with suspected CSD and compared it to that of IFA. From March 2011 to May 2016, at the Microbiology and Virology Unit, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy, 115 clinical specimens (56 aspirated pus, 39 fresh lymph node biopsies, and 20 whole blood samples) and 99 sera from 115 patients with suspected CSD (62 females and 53 males between the ages of 3 months and 68 years) were analyzed by both real-time PCR, used in a qualitative way, and IFA (IgM and IgG) for the presence of B. henselae. For 16 patients, serological results were not available due to a clinical decision not to request the test. B. henselae DNA positivity was detected by real-time PCR in 37.39% of patients, while 62.61% of them were negative. Thus, patients were divided into two groups: real-time PCR+ (n = 43) and real-time PCR- (n = 72). Real-time PCR screening of whole blood, biopsies, and aspirated pus revealed B. henselae positivity in 40%, 38.46%, and 35.71% of patients, respectively. When we analyzed samples by IFA, we found the presence of B. henselae in 28 out of 99 (28.28%) patients, of which 11 (11.11%) belonged to the real-time PCR+ group and 17 (17.17%) to the real-time PCR- group. Among the 71 seronegative subjects, 16 (16.16%) were found positive for B. henselae by real-time PCR. Thus, by combining the results of both assays, we were able to increase the percentage of B. henselae positive specimens from 27.27% (real-time PCR) or 28.28% (IFA) to 44.44% (real-time PCR+IFA). Altogether, these findings indicate that the early detection of B. henselae in patients with suspicious CSD through combined real-time PCR and serological analyses can lead to a more accurate diagnosis of CSD, thereby allowing prompt and appropriate disease management.

The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1ß by Human Cytomegalovirus through Inhibition of NF-kB Activity.

Interleukin-1ß (IL-1ß) is a key effector of the inflammasome complex in response to pathogens and danger signals. Although it is well known that assembly of the inflammasome triggers proteolytic cleavage of the biologically inactive precursor pro-IL-1ß into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1ß production via the inflammasome is still poorly understood. Here, we show that the infection of human foreskin fibroblasts (HFFs) with a mutant HCMV lacking the tegument protein pp65 (v65Stop) results in higher expression levels of mature IL-1ß compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1ß. Furthermore, we show that enhanced IL-1ß production by the v65Stop mutant is due in part to induction of DNA binding and the transcriptional activity of NF-κB. Lastly, we demonstrate that HCMV infection of HFFs triggers a non-canonical IL-1ß activation pathway where caspase-8 promotes IL-1ß maturation independently of caspase-1. Altogether, our findings provide novel mechanistic insights into the interplay between HCMV and the inflammasome system and raise the possibility of targeting pp65 to treat HCMV infection.