Extracellular vesicles: Messengers of allergic immune responses and novel therapeutic strategy.

Extracellular vesicles (EVs) are nanosized particles released by nearly every cell type across all kingdoms of life. As a result, EVs are ubiquitously present in various human body fluids. Composed of a lipid bilayer, EVs encapsulate proteins, nucleic acids, and metabolites, thus playing a crucial role in immunity, for example, by enabling intercellular communication. More recently, there has been increasing evidence that EVs can also act as key regulators of allergic immune responses. Their ability to facilitate cell-to-cell contact and to transport a variety of different biomolecules enables active modulation of both innate and adaptive immune processes associated with allergic reactions. A comprehensive understanding of the intricate mechanisms underlying the interactions among allergens, immune cells, and EVs is imperative to develop innovative strategies for controlling allergic responses. This review highlights the recent roles of host cell- and bacteria-derived EVs in allergic diseases, presenting experimental and clinical evidence that underscores their significance. Additionally, the therapeutic potential of EVs in allergy management is outlined, along with the challenges associated with targeted delivery and cargo stability for clinical use. Optimization of EV composition and targeting strategies holds promise for advancing translational applications and establishing EVs as biomarkers or safe therapeutics for assessing allergic reactions. For these reasons, EVs represent a promising avenue for advancing both our understanding and management of allergic immune processes.

Bacterial extracellular vesicles repress the vascular protective factor RNase1 in human lung endothelial cells.

BACKGROUND: Sepsis is one of the leading causes of death worldwide and characterized by blood stream infections associated with a dysregulated host response and endothelial cell (EC) dysfunction. Ribonuclease 1 (RNase1) acts as a protective factor of vascular homeostasis and is known to be repressed by massive and persistent inflammation, associated to the development of vascular pathologies. Bacterial extracellular vesicles (bEVs) are released upon infection and may interact with ECs to mediate EC barrier dysfunction. Here, we investigated the impact of bEVs of sepsis-related pathogens on human EC RNase1 regulation. METHODS: bEVs from sepsis-associated bacteria were isolated via ultrafiltration and size exclusion chromatography and used for stimulation of human lung microvascular ECs combined with and without signaling pathway inhibitor treatments. RESULTS: bEVs from Escherichia coli, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium significantly reduced RNase1 mRNA and protein expression and activated ECs, while TLR2-inducing bEVs from Streptococcus pneumoniae did not. These effects were mediated via LPS-dependent TLR4 signaling cascades as they could be blocked by Polymyxin B. Additionally, LPS-free ClearColi™ had no impact on RNase1. Further characterization of TLR4 downstream pathways involving NF-кB and p38, as well as JAK1/STAT1 signaling, revealed that RNase1 mRNA regulation is mediated via a p38-dependent mechanism. CONCLUSION: Blood stream bEVs from gram-negative, sepsis-associated bacteria reduce the vascular protective factor RNase1, opening new avenues for therapeutical intervention of EC dysfunction via promotion of RNase1 integrity. Video Abstract.

Bacterial vesicles block viral replication in macrophages via TLR4-TRIF-axis.

Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella, E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella, E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. Video Abstract.

Surface proteome of plasma extracellular vesicles as mechanistic and clinical biomarkers for malaria.

PURPOSE: Malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, mainly in tropical and subtropical countries. Plasmodium falciparum (P. falciparum) is the most prevalent cause on the African continent and responsible for most malaria-related deaths globally. Important medical needs are biomarkers for disease severity or disease outcome. A potential source of easily accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an EV Array to find proteins on plasma sEVs that are differentially expressed in malaria patients. Plasma samples from 21 healthy subjects and 15 malaria patients were analyzed. The EV array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy subjects and malaria patients. We found CD106 to be the best discrimination marker based on receiver operating characteristic (ROC) analysis with an area under the curve of > 0.974. Additional ensemble feature selection revealed CD106, Osteopontin, CD81, major histocompatibility complex class II DR (HLA-DR), and heparin binding EGF like growth factor (HBEGF) together with thrombocytes to be a feature panel for discrimination between healthy and malaria. TNF-R-II correlated with HLA-A/B/C as well as CD9 with CD81, whereas Osteopontin negatively correlated with CD81 and CD9. Pathway analysis linked the herein identified proteins to IFN-γ signaling. CONCLUSION: sEV-associated proteins can discriminate between healthy individuals and malaria patients and are candidates for future predictive biomarkers. TRIAL REGISTRATION: The trial was registered in the Deutsches Register Klinischer Studien (DRKS-ID: DRKS00012518).

Evaluating a multimodal, clinical and work-directed intervention (RTW-PIA) to support sustainable return to work among employees with mental disorders: study protocol of a multicentre, randomised controlled trial.

BACKGROUND: Mental disorders (MDs) are one of the leading causes for workforce sickness absence and disability worldwide. The burden, costs and challenges are enormous for the individuals concerned, employers and society at large. Although most MDs are characterised by a high risk of relapse after treatment or by chronic courses, interventions that link medical-psychotherapeutic approaches with work-directed components to facilitate a sustainable return to work (RTW) are rare. This protocol describes the design of a study to evaluate the (cost-)effectiveness and implementation process of a multimodal, clinical and work-directed intervention, called RTW-PIA, aimed at employees with MDs to achieve sustainable RTW in Germany. METHODS: The study consists of an effectiveness, a health-economic and a process evaluation, designed as a two-armed, multicentre, randomised controlled trial, conducted in German psychiatric outpatient clinics. Sick-listed employees with MDs will receive either the 18-month RTW-PIA treatment in conjunction with care as usual, or care as usual only. RTW-PIA consists of a face-to-face individual RTW support, RTW aftercare group meetings, and web-based aftercare. Assessments will be conducted at baseline and 6, 12, 18 and 24 months after completion of baseline survey. The primary outcome is the employees´ achievement of sustainable RTW, defined as reporting less than six weeks of working days missed out due to sickness absence within 12 months after first RTW. Secondary outcomes include health-related quality of life, mental functioning, RTW self-efficacy, overall job satisfaction, severity of mental illness and work ability. The health-economic evaluation will be conducted from a societal and public health care perspective, as well as from the employer's perspective in a cost-benefit analysis. The design will be supplemented by a qualitative effect evaluation using pre- and post-interviews, and a multimethod process evaluation examining various predefined key process indicators from different stakeholder perspectives. DISCUSSION: By applying a comprehensive, multimethodological evaluation design, this study captures various facets of RTW-PIA. In case of promising results for sustainable RTW, RTW-PIA may be integrated into standard care within German psychiatric outpatient clinics. TRIAL REGISTRATION: The study was prospectively registered with the German Clinical Trials Register ( DRKS00026232 , 1 September 2021).

Surface Proteome of Plasma Extracellular Vesicles as Biomarkers for Pneumonia and Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Community-acquired pneumonia (CAP) and acute exacerbation of chronic obstructive pulmonary disease (AECOPD) represent a major burden of disease and death and their differential diagnosis is critical. A potential source of relevant accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an extracellular vesicle array to find proteins on plasma sEVs that are differentially expressed and possibly allow the differential diagnosis between CAP and AECOPD. Plasma samples were analyzed from 21 healthy controls, 24 patients with CAP, and 10 with AECOPD . The array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy controls and patients with CAP or AECOPD. We found CD45 and CD28 to be the best discrimination markers between CAP and AECOPD in receiver operating characteristic analyses, with an area under the curve >0.92. Additional ensemble feature selection revealed the possibility to distinguish between CAP and AECOPD even if the patient with CAP had COPD, with a panel of CD45, CD28, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), tumor necrosis factor-R-II, and CD16. CONCLUSION: The discrimination of sEV-associated proteins is a minimally invasive method with potential to discriminate between CAP and AECOPD.

Proviral MicroRNAs Detected in Extracellular Vesicles From Bronchoalveolar Lavage Fluid of Patients With Influenza Virus-Induced Acute Respiratory Distress Syndrome.

Influenza A virus (IAV) causes severe respiratory infections and alveolar epithelial damage resulting in acute respiratory distress syndrome (ARDS). Extracellular vesicles (EVs) have been shown to mediate cellular crosstalk in inflammation by transfer of microRNAs (miRNAs). In this study, we found significant changes in the miRNA composition of EVs in the bronchoalveolar lavage fluid from patients with IAV-induced ARDS. Among the 9 significantly deregulated microRNAs, miR-17-5p was upregulated in patients' BALF and in EVs of IAV-infected lung epithelial cells (A549). In these cells, transfer of miR-17-5p strongly downregulated expression of the antiviral factor Mx1 and significantly enhanced IAV replication.

Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.

Dermatologische Aspekte aus der S2k-Leitlinie zum Down-Syndrom im Kindes- und Jugendalter.

Das Down-Syndrom (DS) ist mit einer Inzidenz von 1 : 700 aller Geburten nicht selten und mit diversen Erkrankungen unterschiedlicher Organsysteme assoziiert. Zu den schwerwiegenden Erkrankungen zählen Herzfehler und Leukämie. Letztere zeigt sich im Neugeborenenalter und geht nicht immer in eine klassische myeloische Leukämie über (transiente myeloproliferative Erkrankung). Dermatologisch bilden die Neugeborenen Pusteln/Vesikulopusteln, die bei DS nicht nur an typische Neugeborenenexantheme und Infektionen, sondern auch an die transiente myeloproliferative Erkrankung denken lassen sollten. Die meisten Dermatosen jedoch sind benigner Natur und umfassen im Wesentlichen Verhornungsstörungen, die sich als Xerosis, Keratosis pilaris, Lichenifikation und Ichthyosis vulgaris zeigen. Typisch, aber nicht spezifisch, ist auch die palmare Vierfingerfurche. Die Patienten neigen häufig zu Follikulitiden, die aufgrund einer Elastolyse in eine Anetodermie übergehen. Die bekannte Immundysbalance erklärt das Auftreten von Autoimmunerkrankungen, die sich an der Haut als Alopecia areata und Vitiligo manifestieren. Als typische Hautveränderungen bei DS zählen zudem Elastosis perforans serpiginosa, Syringome, Milien-ähnliche Calcinosis cutis (Milia-like calcinosis cutis) und Multiple eruptive Dermatofibrome.

Dermatological aspects of the S2k guidelines on Down syndrome in childhood and adolescence.

With an incidence of 1 in 700 births, Down syndrome (DS) is not an uncommon condition. It is associated with various disorders of different organ systems. Serious disorders include cardiac defects and leukemia. With an onset during the newborn period, the latter does not always progress to classic myeloid leukemia (transient myeloproliferative disorder). Skin manifestations in newborns include pustules/vesiculopustules. In individuals with DS, such lesions should not only prompt suspicion for typical neonatal rashes and infections but also for transient myeloproliferative disorder. However, most dermatoses are benign. They essentially comprise disorders of keratinization that present as xerosis, keratosis pilaris, lichenification, and ichthyosis vulgaris. Also typical but not specific is the four-finger palmar crease (simian crease). Patients frequently develop folliculitides, which - due to elastolysis - subsequently progress to anetoderma. The known immune disturbance in DS patients explains the occurrence of autoimmune diseases such as alopecia areata and vitiligo. Typical skin conditions associated with DS include elastosis perforans serpiginosa, syringomas, milia-like calcinosis cutis, and multiple eruptive dermatofibromas.

Genetic Polymorphisms as Predictive Markers of Response to Growth Hormone Therapy in Children with Growth Hormone Deficiency.

Objective Growth hormone (GH) deficiency (GHD) is commonly treated with recombinant human GH (rhGH). Individual response to rhGH therapy varies widely and there is evidence that variations in growth-related genes, e. g. the GH receptor (GHR) gene, may impact treatment response. We aimed to identify genetic polymorphisms which could serve as predictive markers of response to rhGH therapy. Methods We conducted a genetic analysis of single nucleotide polymorphisms (SNPs) and the GHR exon 3 deletion in 101 paediatric GHD patients receiving rhGH. Patients were analysed for 13 known SNPs in 11 genes of the GH axis (SOS1, IGFR1, GAB1, LHX4, IGFBP3, GRB10, GHRHR, GHSR), growth plate (VDR, ESR1) and cell cycle (CDK4). Individual index of responsiveness (IoR) values were compared by genotype. We also analysed the potential association between the IoR and the GHR exon 3 deletion. IoRs were analysed by genotype by one-way analysis of variance and unpaired t-test. Results Variations in two SNPs, rs2888586 (SOS1) and rs2069502 (CDK4), and the GHR exon 3 deletion were significantly associated with response to rhGH treatment. Conclusions Genetic variations are potentially suitable as predictive markers of rhGH treatment response in GHD. Genetic analysis provides a starting point for individualised treatment of GHD.

Thermographic imaging in sports and exercise medicine: A Delphi study and consensus statement on the measurement of human skin temperature.

The importance of using infrared thermography (IRT) to assess skin temperature (tsk) is increasing in clinical settings. Recently, its use has been increasing in sports and exercise medicine; however, no consensus guideline exists to address the methods for collecting data in such situations. The aim of this study was to develop a checklist for the collection of tsk using IRT in sports and exercise medicine. We carried out a Delphi study to set a checklist based on consensus agreement from leading experts in the field. Panelists (n = 24) representing the areas of sport science (n = 8; 33%), physiology (n = 7; 29%), physiotherapy (n = 3; 13%) and medicine (n = 6; 25%), from 13 different countries completed the Delphi process. An initial list of 16 points was proposed which was rated and commented on by panelists in three rounds of anonymous surveys following a standard Delphi procedure. The panel reached consensus on 15 items which encompassed the participants' demographic information, camera/room or environment setup and recording/analysis of tsk using IRT. The results of the Delphi produced the checklist entitled "Thermographic Imaging in Sports and Exercise Medicine (TISEM)" which is a proposal to standardize the collection and analysis of tsk data using IRT. It is intended that the TISEM can also be applied to evaluate bias in thermographic studies and to guide practitioners in the use of this technique.

Absence of inferior vena cava in 14-year old boy associated with deep venous thrombosis and positive Mycoplasma pneumoniae serum antibodies--a case report.

BACKGROUND: Absence of the inferior vena cava is a rare vascular anomaly, which usually remains asymptomatic in childhood. It is recognized as the risk factor for deep venous thrombosis, since the collateral circulation does not provide adequate drainage of the lower limbs. Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in school-aged children and adolescents. Mycoplasma pneumoniae infection might be associated with deep venous thrombosis but its pathophysiology remains unknown. According to previous reports, deep venous thrombosis due to Mycoplasma pneumoniae infection is associated with positive serum anticardiolipin antibodies. To our knowledge, we describe the first case of deep venous thrombosis associated with Mycoplasma pneumoniae serum antibodies indicating early stage of infection with negative anticardiolipin serum antibodies in adolescent with absence of inferior vena cava. CASE PRESENTATION: 14-year old boy was admitted to the pediatric unit few days after the appendectomy complaining with pain of the left hip that caused him unable to walk. The pain was accompanied with subfebrile temperature. After clinical examination and additional tests, the boy was diagnosed with a deep venous thrombosis. Computed tomography revealed absence of the vena cava inferior distally to the hepatic veins and varices of the collateral circulation in the pelvis. Anticardiolipin IgM and IgG antibodies and antinuclear antibodies were not detected. Additionally, the Mycoplasma pneumoniae antibodies in classes IgM, IgA and IgG were detected in serum as another risk factor of thrombosis. After the initial treatment with low-molecular-weight heparin in combination with clarithromycin the clinical condition of the patient improved. The patient became a candidate for life-long anticoagulation therapy. CONCLUSIONS: In this case Mycoplasma pneumoniae antibodies were associated with deep venous thrombosis in child with congenital absence of inferior vena cava. Uncommonly for deep venous thrombosis due to Mycoplasma pneumoniae infection, anticardiolipin antibodies were not detected in serum. It is important to remember in clinical practice that Mycoplasma pneumoniae affects coagulability and may trigger thrombosis, especially in the presence of other risk factors. The pathophysiology of this process remains unknown.

Thermal imaging evaluation of paravertebral block for mastectomy in high risk patient: case report.

Thoracic paravertebral block is the technique of injecting local anesthetic adjacent to the thoracic vertebra close to where the spinal nerves emerge from the intervertebral foramina. It is effective in treating acute and chronic pain of unilateral origin from the chest and abdomen. This technique causes pain relief with pulmonary function preservation and great hemodynamic stability. 66 year old woman (156 cm, 80 kg, BMI 32) with chronic right heart failure, hypertension and obesity, on chronic oxygen therapy was presented for elective mastectomy due to breast cancer. She suffered from severe COPD and also bullous emphysema. FVC 1.59 l; FEV1 0.55 l; FEV1%FVC 34.6. The paravertebral block was performed using the multi-shot percutaneous technique with additional light general anesthesia. For confirmation, of proper analgesia range, control of temperature changes, using FLIR i7 infrared camera, was performed. Control photos were made 20 min after the blockade and then 10 min later. Infrared photo showed rise of temperature reading in every marked region. There were no hemodynamic and pulmonary complications postoperatively. Paravertebral block in combination with sedation creates excellent conditions for breast surgery procedures. Additional temperature changes monitoring performed with infrared camera may confirm proper range of analgesia needed to perform surgery. Great cardiovascular stability and very good pulmonary function preservation make this method excellent for high risk patients. Low complication rate is additional advantage. In our opinion this method is recommendable.

Interleukin 18 and neutrophil-gelatinase associated lipocalin in assessment of the risk of contrast-induced nephropathy in children.

The aim of the study was to determine the usefulness of interleukin 18 (IL-18) and neutrophil-gelatinase associated lipocalin (NGAL) in the risk assessment of contrast nephropathy in children. The study included patients among whom radiological examinations were performed using intravascular contrast agent. The material consisted of 33 children (19 girls, 14 boys) aged 6.37 ±5.41 years. 20/33 (61%) of patients had hydronephrosis, 9/33 (27%) had other urinary tract defects referred as "no hydronephrosis" and 4/33 (12%) had urolithiasis. NGAL determination was performed with the use of Human Lipocalin-2 / NGAL Immunoassay. To determine the concentration of human IL-18 an ELISA Kit (MBL International Corporation) was used. There were no statistically significant differences in the concentrations of NGAL and IL-18 in serum determined before the procedure, and after the administration of contrast agent. Concentrations of NGAL and IL-18 were determined in urine three times: before the procedure, 2-4 hours after administration of the contrast agent, and 48 hours after the performed procedure. The analysis showed that the concentration of IL-18 and NGAL in urine did not differ significantly in three consecutive preformed measurements. The study has also found no statistically significant differences between serum creatinine before and 48 hours after injection of contrast. Implementation of new biomarkers such as NGAL and IL-18 expands the possibilities of renal function assessment in children undergoing radiological procedures using contrast agents. In examined children with normal or slightly impaired renal function they did not demonstrate the risk of contrast nephropathy.

Interleukin-18 and NGAL in assessment of ESWL treatment safety in children with urolithiasis.

Urolithiasis is recurrent chronic disease and a complex nephro-urological problem. Currently it is diagnosed in very young children, even infants in the first quarter of life. Until recently the main method of treatment for stones, which for various reasons did not pass spontaneously, was open surgery. At present, the main method replacing open surgery is extracorporeal shock wave lithotripsy (ESWL). Usefulness of common known indicators of the renal function to assess the safety of ESWL procedure is evaluated and verified. The basic markers are serum creatinine, cystatin C, urea, glomerular filtration rate and albuminuria assessment. Unfortunately all these methods show little sensitivity in the case of acute injury processes. There are efforts to use new biomarkers of renal tubular activity, which include among others interleukin 18 (IL-18) and neutrophil gelatinase-associated lipocalin (NGAL). The aim of the study was to assess the safety of ESWL by means of albumin to creatinine ratio, serum cystatin C levels and concentration of two new markers: IL -18 and NGAL. Albumin to creatinine ratio (p = 0.28) and serum cystatin C (p = 0.63) collected before and 48 hours after ESWL did not show statistically significant differences. Similarly, both new markers (IL -18 and NGAL) showed no significant differences (urine IL -18 p = 0.31; serum NGAL p = 0.11; urine NGAL p = 0.29). In conclusion, serum cystatin C tests, urine albumin to creatinine ratio and new early markers of renal tubular injury confirmed the safety of the extracorporeal shock wave lithotripsy (ESWL) and show that the procedure does not cause any episode of acute renal injury.

Does Osteopathic Heart-Focused Palpation Modify Heart Rate Variability in Stressed Participants with Musculoskeletal Pain? A Randomised Controlled Pilot Study.

Heart rate variability (HRV) describes fluctuations in time intervals between heartbeats and reflects autonomic activity. HRV is reduced in stressed patients with musculoskeletal pain and improved after osteopathic manipulative treatment and mind-body interventions. Heart-focused palpation (HFP) combines manual and mind-body approaches to facilitate relaxation. This randomised controlled pilot study investigated the feasibility and sample size for a future randomised controlled trial and the effect of a single treatment with HFP or sham HFP (SHAM) on short-term HRV. A total of Thirty-three adults (47.7 ± 13.5 years old) with stress and musculoskeletal pain completed the trial with acceptable rates of recruitment (8.25 subjects per site/month), retention (100%), adherence (100%), and adverse events (0%). HFP (n = 18), but not SHAM (n = 15), significantly increased the root mean square of successive RR interval differences (p = 0.036), standard deviation of the NN intervals (p = 0.009), and ratio of the low-frequency to high-frequency power band (p = 0.026). HFP and SHAM significantly decreased the heart rate (p < 0.001, p = 0.009) but not the stress index and ratio of the Poincaré plot standard deviation along and perpendicular to the line of identity (p > 0.05). A power analysis calculated 72 participants. Taken together, the study was feasible and HFP improved HRV in stressed subjects with musculoskeletal pain, suggesting a parasympathetic effect.

Lipid A in outer membrane vesicles shields bacteria from polymyxins.

The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae's antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.

Expression of TMPRSS2 is up-regulated by bacterial flagellin, LPS, and Pam3Cys in human airway cells.

Many viruses require proteolytic activation of their envelope proteins for infectivity, and relevant host proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of influenza A virus (IAV) and various coronaviruses (CoV). Increased TMPRSS2 expression has been associated with a higher risk of severe influenza infection and enhanced susceptibility to SARS-CoV-2. Here, we found that Legionella pneumophila stimulates the increased expression of TMPRSS2-mRNA in Calu-3 human airway cells. We identified flagellin as the dominant structural component inducing TMPRSS2 expression. The flagellin-induced increase was not observed at this magnitude for other virus-activating host proteases. TMPRSS2-mRNA expression was also significantly increased by LPS, Pam3Cys, and Streptococcus pneumoniae, although less pronounced. Multicycle replication of H1N1pdm and H3N2 IAV but not SARS-CoV-2 and SARS-CoV was enhanced by flagellin treatment. Our data suggest that bacteria, particularly flagellated bacteria, up-regulate the expression of TMPRSS2 in human airway cells and, thereby, may support enhanced activation and replication of IAV upon co-infections. In addition, our data indicate a physiological role of TMPRSS2 in antimicrobial host response.

Novel protein biomarkers for pneumonia and acute exacerbations in COPD: a pilot study.

Introduction: Community-acquired pneumonia (CAP) and acute exacerbations of chronic obstructive pulmonary disease (AECOPD) result in high morbidity, mortality, and socio-economic burden. The usage of easily accessible biomarkers informing on disease entity, severity, prognosis, and pathophysiological endotypes is limited in clinical practice. Here, we have analyzed selected plasma markers for their value in differential diagnosis and severity grading in a clinical cohort. Methods: A pilot cohort of hospitalized patients suffering from CAP (n = 27), AECOPD (n = 10), and healthy subjects (n = 22) were characterized clinically. Clinical scores (PSI, CURB, CRB65, GOLD I-IV, and GOLD ABCD) were obtained, and interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-2-receptor (IL-2R), lipopolysaccharide-binding protein (LBP), resistin, thrombospondin-1 (TSP-1), lactotransferrin (LTF), neutrophil gelatinase-associated lipocalin (NGAL), neutrophil-elastase-2 (ELA2), hepatocyte growth factor (HGF), soluble Fas (sFas), as well as TNF-related apoptosis-inducing ligand (TRAIL) were measured in plasma. Results: In CAP patients and healthy volunteers, we found significantly different levels of ELA2, HGF, IL-2R, IL-6, IL-8, LBP, resistin, LTF, and TRAIL. The panel of LBP, sFas, and TRAIL could discriminate between uncomplicated and severe CAP. AECOPD patients showed significantly different levels of LTF and TRAIL compared to healthy subjects. Ensemble feature selection revealed that CAP and AECOPD can be discriminated by IL-6, resistin, together with IL-2R. These factors even allow the differentiation between COPD patients suffering from an exacerbation or pneumonia. Discussion: Taken together, we identified immune mediators in patient plasma that provide information on differential diagnosis and disease severity and can therefore serve as biomarkers. Further studies are required for validation in bigger cohorts.