COVID-19: Development and implementation of a video-conference-based educational concept to improve the hygiene skills of health and nursing professionals in the Republic of Kosovo.

Introduction: The COVID-19 pandemic caught the health care systems of all countries unprepared. For the further education of healthcare personnel in the Republic of Kosovo, it became necessary to implement a concept for practical training in hygienic working. A video-conference-based educational concept to bridge the physical distance between Germany and Kosovo enabled the rapid, theoretical and practical transfer of knowledge. Methods: Current evidence on COVID-19 and Standard Operation Procedures (SOP) were researched. Healthcare staff in Pristina were advised and trained in ten online sessions on hygienic working under pandemic conditions via the "Logitech Rally for DFNconf" video conferencing system. The seminars were interpreted consecutively (Albanian). The Situational Judgement Test (HygiKo-SJT) should describe changes in participants' hygiene knowledge. Results: A total of 25 people were trained in hygiene-related knowledge in terms of basic and specific hygiene for COVID-19. The weekly training sessions made it possible to address questions and subsequently provide practical knowledge. The HygiKo-SJT did not show a generalizable, measurable improvement in hygiene competence. Conclusion: Participants benefited from the concept and rapid implementation of a live video-conference-based seminar on "Hygiene under Pandemic Conditions". The positive experience in terms of guidance, advice and training provides the basis for implementing a dedicated "Hygiene" module in Kosovo.

[Reduction of Visitor- and Staff-Associated Risk of Infection by Complex Intervention in the Department of Feto-Maternal Medicine]. / Reduktion des Besucher- und Personal- assoziierten Infektionsrisikos durch komplexe Intervention auf perinatologischen Stationen.

AIM: Clinical trial registration of this trial: is to demonstrate in a department of feto-maternal medicine: (a) that a complex intervention improves hand hygiene of visitors, siblings and staff; and (b) that automated voice prompts at disinfectant dispensers improve rate of hand disinfection. STUDY DESIGN: (a) Pre-/post-test follow-up design with control (1-12/2016), intervention (1-12/2017), follow-up-period (1/2018-12/2019); and (b) RCT in quasi crossover design. Primary endpoints: (a) disinfectant consumption (DC) per patient-day, and (b) DC at disinfectant dispenser per passer-by. RESULTS: A multimodal strategy within the intervention period showed a relevant positive effect on hand hygiene compliance (in IP: 26.2% more DC; p=0.088). Voice prompts increased DC by 28.6% (p=0.025). The odds ratio for high positive fingertip testing plates of visiting children (siblings) between control and intervention period was 0.35 (95% CI [0.1074-0.9708] p=0.039). CONCLUSION: Complex intervention and electronic voice prompts on disinfectant dispensers improve hand hygiene behaviour in perinatology. Installation of disinfectant dispensers in a child-friendly position and adequate information material appeal to children. The data represent an important contribution to improve hand hygiene of visitors, siblings and staff in hospitals in a pandemic situation.

Neutrophil elastase-mediated increase in airway temperature during inflammation.

BACKGROUND: How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection. METHODS: We determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq. RESULTS: Here we show a temperature of ~38°C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38°C vs 30°C revealed increased virulence traits and characteristic cell wall changes. CONCLUSION: Neutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF.

SFTA3, a novel protein of the lung: three-dimensional structure, characterisation and immune activation.

The lung constantly interacts with numerous pathogens. Thus, complex local immune defence mechanisms are essential to recognise and dispose of these intruders. This work describes the detection, characterisation and three-dimensional structure of a novel protein of the lung (surfactant-associated protein 3 (SFTA3/SP-H)) with putative immunological features. Bioinformatics, biochemical and immunological methods were combined to elucidate the structure and function of SFTA3. The tissue-specific detection and characterisation was performed by using electron microscopy as well as fluorescence imaging. Three-dimensional structure generation and analysis led to the development of specific antibodies and, as a consequence, to the localisation of a novel protein in human lung under consideration of cystic fibrosis, asthma and sepsis. In vitro experiments revealed that lipopolysaccharide induces expression of SFTA3 in the human lung alveolar type II cell line A549. By contrast, the inflammatory cytokines interleukin (IL)-1ß and IL-23 inhibit expression of SFTA3 in A549. Sequence- and structure-based prediction analysis indicated that the novel protein is likely to belong to the family of lung surfactant proteins. The results suggest that SFTA3 is an immunoregulatory protein of the lung with relevant protective functions during inflammation at the mucosal sites.

Staphylococcus aureus and Pseudomonas aeruginosa express and secrete human surfactant proteins.

Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express 'human-like' surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment.

Expression and regulation of antimicrobial peptide psoriasin (S100A7) at the ocular surface and in the lacrimal apparatus.

PURPOSE: Psoriasin, originally isolated from psoriasis as an overexpressed molecule of unknown function, has recently been identified as a principal Escherichia coli-killing antimicrobial peptide of healthy skin. The purpose of this study was to investigate the expression and antimicrobial role of psoriasin at the ocular surface and in the lacrimal apparatus. METHODS: Different tissues of the lacrimal apparatus and ocular surface were systematically analyzed by means of RT-PCR, Western blot, and immunohistochemistry for their ability to express and produce psoriasin. The inducibility and regulation of psoriasin were studied in human corneal as well as conjunctival epithelial cell lines after challenge with ocular pathogens and proinflammatory cytokines. Real-time RT-PCR was performed to evaluate the expression and induction of psoriasin. In addition, tear fluid obtained from different healthy volunteers was examined by ELISA for its psoriasin concentration. RESULTS: RT-PCR and Western blot analyses revealed a constitutive expression of psoriasin in cornea, conjunctiva, nasolacrimal ducts, and lacrimal gland. Immunohistochemistry showed strong staining of meibomian glands for psoriasin. No induction of psoriasin was observed after stimulation with supernatants of E. coli, whereas supernatants of Staphylococcus aureus and Haemophilus influenzae significantly increased the psoriasin mRNA expression. Stimulation with IL-1ß and VEGF also strongly increased psoriasin transcription. The highest amounts of psoriasin protein were detected in the tear fluid (~170 ng/mL) of healthy volunteers. CONCLUSIONS: The results suggest that psoriasin is produced by the structures of the ocular surface and is part of the innate immune system at the ocular surface and tear film.

Lactate in cystic fibrosis sputum.

BACKGROUND: Antibiotic therapy is thought to improve lung function in patients with cystic fibrosis (CF) by decreasing neutrophil-derived inflammation. We investigated the origin and clinical significance of lactate in the chronically inflamed CF lung. METHODS: Lactate was measured in sputa of 18 exacerbated and 25 stable CF patients via spectrophotometry and gaschromatography. Lung function was assessed via spirometry. Seven patients with chronic obstructive pulmonary disease (COPD) and three patients with acute lung inflammation served as control groups. Neutrophil and bacterial lactate production was assessed under aerobic and anaerobic conditions. RESULTS: In sputum specimens of patients with respiratory exacerbations lactate concentrations decreased significantly (p<0.005) from 3.4±2.3mmol/L to 1.4±1.4mmol/L after 2-3 weeks of intravenous antibiotics. Successful treatment was reflected in 16 patients (88.9%) by FVC increase associated with lactate decrease (p<0.05). In every single sputum lactate was detectable (3.0±3.1mmol/L, range 0.2-14.1mmol/L). Lactate was lower (1.6±0.8mmol/L) in sputa from seven COPD patients, and it was below the detection limit in three patients with acute lung inflammation. Neutrophil lactate production accumulated up to 10.5mmol/L after 4 days, whereas bacterial lactate production did not appear to contribute substantially to sputum lactate concentrations. CONCLUSIONS: Successful antibiotic therapy is reflected by a decrease in lactate concentrations. Neutrophils are the most likely source for lactate in sputum of CF patients. Therefore lactate may be used to monitor responses to antibiotic therapy as an adjunct to lung function measurements.

Roles of human beta-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells.

Human beta-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of beta-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human beta-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse beta-defensins-2, -3 and -4 (mBD2-4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of beta-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1beta is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that beta-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of beta-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of beta-defensins at the ocular surface and lacrimal apparatus and show how beta-defensins are regulated specifically.

Alveolar inflammation in cystic fibrosis.

BACKGROUND: In infected lungs of the cystic fibrosis (CF) patients, opportunistic pathogens and mutated cystic fibrosis transmembrane conductance regulator protein (CFTR) contribute to chronic airway inflammation that is characterized by neutrophil/macrophage infiltration, cytokine release and ceramide accumulation. We sought to investigate CF lung inflammation in the alveoli. METHODS: Lung tissue from 14 CF patients and four healthy individuals was analyzed for numbers of effector cells, elastin and collagen concentrations, inflammatory markers and density of Pseudomonas aeruginosa. Additionally, desmosine and isodesmosine concentrations were determined in 52 urine specimens from CF patients to estimate the burden of elastase activities in respiratory secretions. RESULTS: Elastin concentration was significantly decreased and collagen significantly increased in CF alveolar tissues as compared to age-matched, healthy individuals. Elastin split products were significantly increased in urine samples from patients with CF and correlated inversely with age, indicating local tissue remodelling due to elastin degradation by unopposed proteolytic enzymes. Alveolar inflammation was also characterized by a significant cell infiltration of neutrophils, macrophages and T cells, extensive nuclear factor-kappaB and insulin-like growth factor-1 activation in various cell types and increased intercellular adhesion molecule-1 expression, and increased numbers of myofibroblasts. Additionally, ceramide accumulated in type II alveolar epithelial cells, lacking CFTR. P. aeruginosa organisms were rarely present in inflamed alveoli. CONCLUSIONS: Chronic inflammation and remodeling is present in alveolar tissues of the CF lung and needs to be addressed by anti-inflammatory therapies.

Detection and regulation of cationic amino acid transporters in healthy and diseased ocular surface.

PURPOSE: To evaluate the presence and role of human cationic amino acid transporters (hCATs) at the ocular surface in healthy and pathologic states and under experimental inflammatory conditions. METHODS: Expression of mRNA for hCATs 1, 2, and 3 (SLC7A1, SLC7A2, and SLC7A3) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts and in an SV40 immortalized human corneal epithelial (HCE) cell line. Localization of hCAT1 and hCAT2 was determined by immunohistochemistry in healthy tissues and in sections of different corneal abnormalities, including keratoconus, Fuchs dystrophy, and herpetic keratitis. Cultured corneal epithelial cells were stimulated with proinflammatory cytokines and supernatants of Staphylococcus aureus and Pseudomonas aeruginosa and were analyzed by real-time PCR. RESULTS: Expression of hCAT1 and hCAT2 mRNA, but not of hCAT3 mRNA, was detected in healthy conjunctiva, cornea, and nasolacrimal ducts. Human lacrimal gland revealed only hCAT2 mRNA expression. Immunohistochemistry demonstrated the presence of hCAT1 and hCAT2 in epithelial cells of cornea, conjunctiva, and nasolacrimal ducts. Goblet cells revealed no reactivity. Moreover, hCAT2 was visible in acinar cells of lacrimal gland. No changes in staining reactivity were obtained for hCAT1 in different corneal abnormalities. In contrast, hCAT2 showed increased subjective staining intensity in all corneal abnormalities. Cell culture experiments revealed that TNF-alpha and supernatant of S. aureus increased hCAT1 and hCAT2 expression significantly. Supernatant of P. aeruginosa led to an increase in hCAT2-expression only. CONCLUSIONS: These results show for the first time the distribution of hCATs in tissues of the ocular surface and lacrimal apparatus. Both transporters seem to be differently regulated under pathologic conditions of the ocular surface. Their physiological functions in amino acid transport make them potential candidates for therapeutic intervention.

Regulation of MUC16 by inflammatory mediators in ocular surface epithelial cell lines.

The aim of the present study was to evaluate the regulation of membrane-anchored mucin MUC16 by proinflammatory cytokines and bacterial components at the ocular surface. Expression and distribution of MUC16 in conjunctival (HCjE) and corneal (HCE) epithelial cell lines was monitored by RT-PCR and immunohistochemistry. To determine the regulation of MUC16, cultured HCjEs and HCEs were stimulated with different cytokines, bacterial components and bacterial supernatants, and analyzed by real-time PCR, immunodot blot and immunohistochemistry. The results indicate that MUC16 is differentially regulated between HCjEs and HCEs after challenge with inflammatory mediators and suggest shedding of MUC16 from the ocular surface epithelia into the tear film. This seems to be precisely regulated. MUC16 shedding can be differentially increased and decreased, suggesting a protective function of membrane-anchored MUC16 and supporting the hypothesis that dysregulation of membrane-anchored MUC16 at the ocular surface may be involved in dry eye pathology.

Virulence of Burkholderia cepacia complex strains in gp91phox-/- mice.

In cystic fibrosis (CF), infection with Burkholderia cepacia complex (Bcc) strains may cause long-term asymptomatic airway colonization, or severe lung infection leading to rapid pulmonary decline. To assess the virulence of Bcc strains, we established a lung infection model in mice with a null allele of the gene involved in X-linked chronic granulomatous disease (CGD). CGD mice, challenged intratracheally with 10(3) cells of the epidemic Burkholderia cenocepacia strain J2315, died within 3 days from sepsis after bacteria had multiplied to 3.3 x 10(8) cells. Infected mice developed neutrophil-dominated lung abscesses. Other B. cenocepacia strains and a B. cepacia strain were less virulent and one B. multivorans and one B. vietnamensis CF isolate were both avirulent. Bcc mutants, defective in exopolysaccharide synthesis or quorum sensing revealed diminished or no abscess formation and mortality. Immunofluorescence staining of Bcc-infected murine and CF lung tissues revealed colocalization of Bcc and neutrophils, suggesting Bcc persistence within neutrophils in CGD and CF. In vitro, Bcc cells were rapidly killed during aerobic neutrophil phagocytosis; however, the pathogens survived in neutrophils with blocked nicotinamide adenine dinucleotide phosphate oxidase activity and under anaerobic conditions. We conclude that the Bcc infection model in CGD mice is well suited for the assessment of Bcc virulence.

Sequence diversity of the mucABD locus in Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The mucA gene of the muc operon, which is instrumental in the control of the biosynthesis of the exopolysaccharide alginate, is a hotspot of mutation in Pseudomonas aeruginosa, a micro-organism that chronically colonizes the airways of individuals with cystic fibrosis (CF). The mucA, mucB and mucD genes were sequenced in nine environmental isolates from aquatic habitats, and in 37 P. aeruginosa strains isolated from 10 patients with CF, at onset or at a late stage of chronic airway colonization, in order to elucidate whether there was any association between mutation and background genotype. The 61 identified single nucleotide polymorphisms (SNPs) segregated into 18 mucABD genotypes. Acquired and de novo stop mucA mutations were present in 14 isolates (38 %) of five mucABD genotypes. DeltaG430 was the most frequent and recurrent mucA mutation detected in four genotypes. The classification of strains by mucABD genotype was generally concordant with that by genome-wide SpeI fragment pattern or multilocus SNP genotypes. The exceptions point to intragenic mosaicism and interclonal recombination as major forces for intraclonal evolution at the mucABD locus.

Nonmucoid Pseudomonas aeruginosa expresses alginate in the lungs of patients with cystic fibrosis and in a mouse model.

BACKGROUND: In patients with cystic fibrosis (CF), lung infection with mucoid Pseudomonas aeruginosa strains overexpressing the exopolysaccaride alginate is preceded by colonization with nonmucoid strains. We investigated the kinetics, impact of environmental signals, and genetics of P. aeruginosa alginate expression in a mouse model and in patients with CF. METHODS: Using indirect immunofluorescence, microarray technology and real-time reverse-transcription polymerase chain reaction, we assessed alginate gene expression during aerobic and anaerobic growth of the nonmucoid strain PAO1 in vitro, in a mouse lung-infection model and in sputum specimens from patients with CF infected with nonmucoid or mucoid P. aeruginosa strains. RESULTS: Anaerobic conditions increased the transcription of alginate genes in vitro and in murine lungs within 24 h. Alginate production by PAO1 in murine lungs and by nonmucoid P. aeruginosa strains in patients with CF was reversible after in vitro culture under aerobic conditions. A subpopulation of P. aeruginosa clones revealing stable alginate production was detected in murine lungs 2 weeks after infection. CONCLUSIONS: Anaerobiosis and lung infection rapidly induce alginate production by gene regulation in nonmucoid P. aeruginosa. This trait may contribute to early persistence, leading to chronic P. aeruginosa infection once stable mucoid strains are generated.

Effect of treatment with dornase alpha on airway inflammation in patients with cystic fibrosis.

Recombinant human deoxyribonuclease (rhDNase) has been shown to improve lung function and reduce the number of pulmonary exacerbations in patients with cystic fibrosis (CF), but its long-term effect on airway inflammation remains unknown. In this study, we used bronchoalveolar lavage (BAL) to investigate the long-term effect of rhDNase on inflammation in patients with CF having mild lung disease. A total of 105 patients with CF (> or =5 years of age) having normal lung function were randomized to receive rhDNase (2.5 mg/day) or no rhDNase. Patients with a normal percentage of neutrophils in BAL fluid at baseline were not randomized and served as the control group. The percentage of neutrophils in the pooled BAL sample was similar in both randomized groups at baseline. A significant increase in neutrophils was observed over the 3-year study period in both untreated patients and control subjects, whereas neutrophils remained unchanged in patients treated with rhDNase. Elastase activities and interleukin-8 concentrations also increased in untreated patients and remained stable in patients on rhDNase. We conclude that in patients with CF, an increase in neutrophilic airway inflammation is found that is positively influenced by rhDNase treatment.

Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.

Current theories of CF pathogenesis predict different predisposing "local environmental" conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Pseudomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O(2) consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.

Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection.

To understand the importance of quorum sensing in chronic Pseudomonas aeruginosa lung infection, the in vivo pathogenic effects of the wild-type P. aeruginosa PAO1 and its double mutant, PAO1 lasI rhlI, in which the signal-generating parts of the quorum sensing systems are defective were compared. The rat model of P. aeruginosa lung infection was used in the present study. The rats were killed on days 3, 7, 14 and 28 after infection with the P. aeruginosa strains. The results showed that during the early stages of infection, the PAO1 double mutant induced a stronger serum antibody response, higher production of pulmonary interferon gamma, and more powerful blood polymorphonuclear leukocyte (PMN) chemiluminescence compared to its wild-type counterpart. On days 14 and 28 post-infection, significantly milder lung pathology, a reduction in the number of mast cells present in the lung foci, a reduced number of lung bacteria, and minor serum IgG and IgG1 responses but increased lung interferon gamma production were detected in the group infected with the PAO1 double mutant when compared with the PAO1-infected group. Delayed immune responses were observed in the PAO1-infected group and they might be associated with the production of virulence factors that are controlled by the quorum sensing systems. The conclusion of this study is that functional lasI and rhlI genes of P. aeruginosa PAO1 play a significant role during lung infection.