Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo.

BACKGROUND: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments. RESULTS: In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsß genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39οC showed a decrease in cellular invasion and an increase in IL-8-but not in IL-6-production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39 °C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37 and 39 °C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39 °C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39 °C comparing with 37 °C. CONCLUSIONS: Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo.

Background: Staphylococcus aureus and Pseudomonas aeruginosa cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39°C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37°C, by using RNAseq and physiological assays including in-vivo experiments. Results: In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes on both species. Altered metabolic features at 39°C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39°C they upregulated ethanol oxidation related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα and pmsß genes at 39°C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphylxantin production and a decrease in biofilm formation at 39°C. On the side of PA monocultures, we observed increase in extracellular lipase and protease and biofilm formation at 39°C along with a decrease in motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in-vitro and in-vivo . S. aureus monocultured at 39°C showed a decrease in cellular invasion and an increase in IL-8 -but not in IL-6- production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39°C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37°C and 39°C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39°C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39°C comparing with 37°C. Conclusion: Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

Aneurysm Treatment in Acute SAH with Hydrophilic-Coated Flow Diverters under Single-Antiplatelet Therapy: A 3-Center Experience.

BACKGROUND AND PURPOSE: In certain clinical circumstances, dual-antiplatelet therapy can be problematic in patients with acute SAH. In some aneurysms, however, flow-diverting stents are the ideal therapeutic option. We report our experience with ruptured intracranial aneurysms treated with flow diverters with hydrophilic coating (p48 MW HPC and p64 MW HPC) under single-antiplatelet therapy. MATERIALS AND METHODS: Patients were treated with either flow-diverter placement alone or a flow diverter and additional coiling. Due to the severity of the hemorrhage, the potential for periprocedural rehemorrhage, and the potential for additional surgical interventions, a single-antiplatelet regimen was used in all patients. RESULTS: Thirteen aneurysms were treated in 10 patients. The median age was 62 years; 5 patients were male. All had acute SAH due to aneurysm rupture. Four blood-blister, 2 dissecting, and 7 berrylike aneurysms were treated. Seven aneurysms were adjunctively coiled. Eight of the 10 patients received a single-antiplatelet protocol of aspirin, 1 patient was treated with prasugrel only, and 1 patient was treated with tirofiban first and then switched to the aspirin single-antiplatelet protocol. One device-related complication occurred, a thrombosis of an overstented branch. All stents, however, remained open at DSA, CTA, or MRA follow-up. CONCLUSIONS: The implantation of flow diverters with reduced thrombogenicity due to hydrophilic surface coating under single-antiplatelet therapy seems to be an option in carefully selected cases of SAH due to aneurysm rupture.

Associated abdominal injuries do not influence quality of care in pelvic fractures-a multicenter cohort study from the German Pelvic Registry.

Background: Pelvic fractures are rare but serious injuries. The influence of a concomitant abdominal trauma on the time point of surgery and the quality of care regarding quality of reduction or the clinical course in pelvic injuries has not been investigated yet. Methods: We retrospectively analyzed the prospective consecutive cohort from the multicenter German Pelvic Registry of the German Trauma Society in the years 2003-2017. Demographic, clinical, and operative parameters were recorded and compared for two groups (isolated pelvic fracture vs. combined abdominal/pelvic trauma). Results: 16.359 patients with pelvic injuries were treated during this period. 21.6% had a concomitant abdominal trauma. The mean age was 61.4 ± 23.5 years. Comparing the two groups, patients with a combination of pelvic and abdominal trauma were significantly younger (47.3 ± 22.0 vs. 70.5 ± 20.4 years; p < 0.001). Both, complication (21.9% vs. 9.9%; p < 0.001) and mortality (8.0% vs. 1.9%; p < 0.001) rates, were significantly higher.In the subgroup of acetabular fractures, the operation time was significantly longer in the group with the combined injury (198 ± 104 vs. 176 ± 81 min, p = 0.001). The grade of successful anatomic reduction of the acetabular fracture did not differ between the two groups. Conclusion: Patients with a pelvic injury have a concomitant abdominal trauma in about 20% of the cases. The clinical course is significantly prolonged in patients with a combined injury, with increased rates of morbidity and mortality. However, the quality of the reduction in the subgroup of acetabular fractures is not influenced by a concomitant abdominal injury. Trial registration: ClinicalTrials.gov, NCT03952026, Registered 16 May 2019, retrospectively registered.

Coiling of wide-necked carotid artery aneurysms assisted by a temporary bridging device (Comaneci): preliminary experience.

BACKGROUND: Endovascular treatment of wide-necked aneurysms remains challenging without the use of adjunctive devices to preserve the parent artery. OBJECTIVE: To present our initial experience with a temporary bridging device, the Comaneci (Rapid Medical, Israel). The compliant remodeling mesh protects the parent artery during coil occlusion without flow arrest in the distal vasculature. Permanent dual antiplatelet therapy is not required since the device is fully removed at the end of the procedure. METHODS: All intracranial aneurysms treated by coil occlusion with the Comaneci device between December 2014 and November 2015 were included. Angiographic and clinical results were retrospectively analysed, including follow-up examinations. All aneurysms were unruptured. RESULTS: 18 aneurysms of the internal carotid artery were included. Successful coil occlusion assisted by the Comaneci device as intended was possible in 14 cases (77.8%). Insufficient coverage of the aneurysmal neck was observed in four cases (22.2%), with modification of the treatment to stent-assisted coiling or remodeling. One clinically relevant complication occurred (5.6%). CONCLUSIONS: Our initial experience shows that use of the Comaneci device is straightforward for the treatment of selected wide-necked aneurysms. Further studies with long-term follow-up data are needed to identify the significance of the presented technique in the neurointerventional armamentarium.

[Not Available]. / Einsatz der CT-Angiographie zur Feststellung des zerebralen Zirkulationsstillstandes.

CT-angiography (CTA) has been accredited as an additional technical method for the detection of the cessation of cerebral blood circulation in the updated German guidelines for the determination of irreversible loss of brain function. A standardized CTA protocol was defined. The evaluation of the CTA has to be done by radiologists with several years of experience in neuroradiology, preferably by radiologists certified as neuroradiologists. The so-called "stasis filling", a slow progressive spread of contrast media into the cerebral arteries despite cessation of cerebral blood circulation, has to be considered.

[The intradermal tuberculin test: literature, directive and implementation in practice]. / Der Tuberkulin-Hauttest: Literatur, Richtlinie und Umsetzung in der Praxis.

OBJECTIVE: Because of an increase in the number of cases of bovine tuberculosis in southern Germany (Allgäu region, mainly in the administrative district Swabia) during recent years, blanket tuberculosis testing was resumed in this region. The aim of this study was to review the veterinarians' current knowledge regarding the technique of the intradermal tuberculin test. As a consequence, a guide with precise instructions for the execution and interpretation of intradermal tuberculin testing in cattle based on the current legislation should be created. MATERIAL AND METHODS: Using a questionnaire, farm-animal practitioners' knowledge and experiences of intradermal tuberculin testing were surveyed, collected and evaluated. Legislative texts on tuberculosis (particularly testing of tuberculosis) were evaluated in their current and previous versions, and compared with the experiences reported by the veterinarians. RESULTS: A total of 137 veterinarians participated and 130 returned questionnaires could be evaluated. Forty-four of the 130 participants were involved in tuberculosis testing when the survey was performed. Of these 44 questionnaires, 42 were incorporated in the final evaluation. The majority of the veterinarians perform the intradermal tuberculosis test as laid down in the Commission Regulation (EC) no. 1226/2002 of 8 July 2002 amending Annex B to Council Directive 64/432/EEC. However, many practitioners do not comply with the requirements of the Commission Regulation (EC) no. 1226/2002 when evaluating the results of the intradermal tuberculosis test. Veterinarians showing the least accordance with required standards only test single animals or work in areas other than Swabia. CONCLUSIONS: In areas severely affected by tuberculosis, the technique of intradermal tuberculosis testing is performed almost as demanded by the Commission Regulation (EC) no. 1226/2002. However, a more uniform and careful approach should be sought when monitoring the results. The guide designed in the context of this study can help to improve the performance of the intradermal tuberculosis test. The information from the literature review also shows that there is currently no standardized method of intradermal tuberculosis testing.

The role of tumour FoxP3 as prognostic marker in different subtypes of head and neck cancer.

Expression of the forkhead transcription factor (FoxP3)--an established marker of regulatory T cells--has been found in other cell types as well, including tumour cells. Recent studies indicated that high tumour FoxP3 expression might be associated with a poor outcome of patients with several types of solid cancers. Here, we investigated the role of FoxP3 expressed by the tumour cells in the prognosis of larynx and oro-hypopharynx squamous cell carcinoma (LSCC and OHSCC)--two major subtypes of head and neck cancer. To this end, we analysed by immunohistochemistry the expression of tumour FoxP3 in tissues from 83 LSCC and 89 OHSCC patients in relation to overall survival. In multivariate analysis we found that high tumour FoxP3 expression significantly associated with poor survival in OHSCC but not in LSCC patients. In further studies, we combined the prognostic value of FoxP3 with selected markers of inflammation (cyclooxygenase-2; COX2) or with markers of enhanced tumour migration/invasion (AHNAK and CORTACTIN). Interestingly, we found that the combination of FoxP3 and AHNAK (in LSCC) or FoxP3 and CORTACTIN (in OHSCC) had significantly stronger prognostic values than either marker analysed individually. Combination of FoxP3 and COX2 enhanced the prognostic accuracy only in OHSCC. Thus, our study identifies novel individual and combination markers that might have enhanced and distinct prognostic relevance in different subtypes of head and neck cancer.

Potent and prolonged hypoglycemic activity of an oral insulin--Tat mixture in diabetic mice.

Trans-activator of transcription (Tat) is a cell penetrating peptide which can translocate and carry macromolecular cargoes through cell membranes. This study investigated the hypoglycemic activity of orally delivered insulin - Tat mixture in alloxan-induced diabetic mice. The mixtures of insulin and Tat at 1:1, 1:3 and 1:6 molar ratios were given orally at the insulin doses ranging from 1-200 IU/kg. The fasting blood glucose (FBG) levels were measured at initial, 1, 2, 4, 6, and 12 h after administration. At 1:3 molar ratio of the mixture and after 12 h of administration, insulin at 200 IU/kg showed the highest with prolonged hypoglycemic activity of 74.0±10.3% FBG reduction (2.18 folds of subcutaneously injected (SC) insulin). Free insulin administered orally did not show any hypoglycemic activity. The mixtures at the insulin doses of 100 and 50 IU/kg also showed potent FBG reduction of 73.8±8.2 and 71.3±16.9% at 12 h after administration (2.18 and 2.10 folds of SC insulin, respectively). After incubation with Mono-Mac-6 cells, only the -mixtures but not the free insulin showed intra-cellular insulin uptake, indicating the insulin penetration through the cell membranes via Tat. In simulated gastric fluid, the insulin content in the mixture was not found, demonstrating the degradation of insulin in the gastric environments. Insulin may be absorbed at upper gastrointestinal tract facilitated by Tat. The potent and prolonged hypoglycemic activity of insulin co-administered orally with Tat can be further developed as an effective oral insulin delivery system.

[The role of CT in the diagnosis of peritonsillar abscesses after Punctio Sicca]. / Stellenwert der Computertomografie in der Peritonsillarabszessdiagnostik nach Punctio sicca.

BACKGROUND: Peritonsillar abscesses are considered to be complications of a peritonsillar inflammation and are a frequently occurring condition. Often, to confirm the presence of an abscess and to locate it following a dry-tap aspirate, computed tomography (CT) is performed. No validated data has been published to date concerning the role of CT in the diagnosis of peritonsillar abscesses. MATERIAL AND METHODS: In a retrospective analysis, the records of patients presenting at a German university hospital with a suspected peritonsillar abscess between 2006 and 2011 were evaluated. A particular focus was placed on the diagnostic procedure for patients with dry tap. The CT images were evaluated by 2 neuroradiologists. RESULTS: 310 patients with a suspected peritonsillar abscess from the period 2006­2011 were evaluated. In 51 cases (31 male and 20 female patients; 16.5 %), a dry tap was found to have occurred. Of these, in 42 patients (82.3 %) a CT scan was performed to rule out or locate an abscess. In 36 patients (85.7 %), an abscess was verified by CT imaging. CONCLUSION: Where there are clinical grounds for suspecting a peritonsillar abscess, and where a dry tap occurs, our data indicate that performing a CT scan to verify and locate an abscess is an effective diagnostic procedure. However, greater attention should be paid to additional diagnostic methods.

Sex-specific effects of spironolactone on blood pressure in gonadectomized male and female Wistar rats.

A low-salt diet is known to decrease and salt excess to increase blood pressure in humans and rodents. Sex steroids seem to play a role in salt dependent hypertension. However, little is known about sex differences in mineralocorticoid receptor blockade between male and female rats. The objective of the work was at first to investigate the effects of a low-salt vs. a high-salt diet on blood pressure without the influence of gonadal steroids in male and female rats. Second, to determine the sex-specific effects of mineralocorticoid receptor blockade by spironolactone in high-salt and low-salt fed gonadectomized male and female animals. Normotensive male and female Wistar rats were gonadectomized and put on a low (NaCl<0.03%) or high (NaCl=4%) salt diet. On each diet animals received spironolactone or placebo. Blood pressure was measured by tail-cuff-method; 24-h urine samples were collected in metabolic cages and blood was collected for hormonal measurements. High-salt diet significantly increased systolic blood pressure in both sexes. This effect could be blocked effectively by spironolactone only in male rats. Spironolactone treatment significantly increased aldosterone levels in males and females independent of the sodium content of the diet. High sodium diet significantly increased relative kidney weight, which was not altered by spironolactone treatment. Independently of gonadal steroids a high-salt diet increased blood pressure in gonadectomized male and female rats. Spironolactone lowered blood pressure only in male not in female rats on a high-salt diet clearly indicating sex-specific effects of the mineralo-corticoid antagonist spironolactone.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in head and neck squamous cell carcinomas.

BACKGROUND: Chronic inflammation plays an important role in head and neck squamous cell carcinomas (HNSCC). This study addresses the impact of two single nucleotide polymorphisms (SNP) Asp299Gly and Thr399Ile of the toll-like receptor (TLR) 4 gene on the clinical outcome while accounting for the influence of adjuvant systemic therapy in a large cohort of HNSCC patients. METHODS: Genotype analysis was done using DNA from tissue samples from 188 patients with HNSCC; TLR4 protein expression was assessed immunohistochemically in tissue microarrays. Classical survival models were used for statistical analyses. RESULTS: Ten percent of patients with HNSCC presented with the TLR4 299Gly and 17% with the TLR4 399Ile allele. Patients with the heterozygous genotype TLR4 Asp299Gly had a significantly reduced disease-free and overall survival. Also, patients with the heterozygous genotype TLR4 Thr399Ile had a reduced disease-free survival. Notably, these associations seem to be attributable to relatively poor therapy response as e.g. reflected in a significantly shorter DFS among HNSCC patients carrying the Asp299Gly variant and receiving adjuvant systemic therapy. CONCLUSION: According to this study, TLR4 299Gly und 399Ile alleles may serve as markers for prognosis of head and neck cancer in patients with adjuvant systemic therapy, particularly chemotherapy, and might indicate therapy resistance.

Polymorphonuclear granulocytes in human head and neck cancer: enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease.

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN-related alterations in HNSCC, the role of tumor-infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN-related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil-to-lymphocyte ratio and serum concentrations of CXCL8 (interleukin-8), CCL4 (MIP-1ß) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC-conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor-derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host-mediated changes in the tumor microenvironment.

An efficient approach for the isolation, identification and evaluation of antimicrobial plant components on an analytical scale, demonstrated by the example of Radix imperatoriae.

Using Radix imperatoriae (the root of masterwort) as an example, we describe an efficient approach for the isolation, identification and evaluation of bioactive plant components on an analytical scale. The extraction of Radix imperatoriae with ethyl acetate was enhanced by the application of ultrasound oscillations. This rhizome extract was applied to three pathogenic bacteria (Bacillus cereus, Escherichia coli, and Staphylococcus aureus) to determine its antimicrobial activity. Disk diffusion was utilized to determine susceptibility. The extract components were separated using a series of chromatography approaches (semi-preparative RP-HPLC, or RP-HPLC on an analytical scale), followed by testing. All fractions were analyzed by LC-UV-ESI-MS and 600 MHz microcoil (1)H NMR spectroscopy. Among other findings, in the fraction with the highest antibacterial activity we were able to identify oxypeucedanin and oxypeucedanin hydrate. Subsequent analysis revealed that only oxypeucedanin hydrate had antibacterial activity, whereas oxypeucedanin itself was inactive at the concentrations applied. Furthermore, oxypeucedanin hydrate appears to be largely, or exclusively, a by-product of sample preparation, since it is either not synthesized by the plant as a second metabolite or is produced by it in only very small quantities.

The Staphylococcus aureus lipoprotein SitC colocalizes with Toll-like receptor 2 (TLR2) in murine keratinocytes and elicits intracellular TLR2 accumulation.

SitC is one of the predominant lipoproteins in Staphylococcus aureus. Recently, SitC was shown to be capable of stimulating Toll-like receptor 2 (TLR2), but the mechanism of TLR2 activation by SitC has not been analyzed in detail so far. In this study, we purified C-terminally His-tagged SitC (SitC-His) from Staphylococcus aureus. SitC-His induced interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release in human monocytes and also NF-κB activation in TLR2-transfected HEK293 cells, indicating TLR2-specific activation. SitC not only induced a TLR2-dependent release of IL-6 in primary murine keratinocytes (MKs) but also induced intracellular accumulation of TLR2, which was time and concentration dependent. Cy2-labeled SitC-His colocalized specifically with TLR2 in MKs and was also internalized in TLR2 knockout MKs, suggesting a TLR2-independent uptake. Neither activation nor colocalization of SitC-His was observed with TLR4 or Nod2. The results show that the native lipoprotein SitC-His specifically colocalizes with TLR2, is internalized by host cells, induces proinflammatory cytokines, and triggers intracellular accumulation of TLR2.