Evaluation of the synergistic effect of eravacycline and tigecycline against carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a substantial healthcare challenge. This study assessed the in vitro efficacy of selected antibiotic combinations against CRKP infections. METHODS: Our research involved the evaluation of 40 clinical isolates of CRKP, with half expressing Klebsiella pneumoniae carbapenemase (KPC) and half producing Metallo-ß-lactamase (MBL), two key enzymes contributing to carbapenem resistance. We determined the minimum inhibitory concentrations (MICs) of four antibiotics: eravacycline, tigecycline, polymyxin-B, and ceftazidime/avibactam. Synergistic interactions between these antibiotic combinations were examined using checkerboard and time-kill analyses. RESULTS: We noted significant differences in the MICs of ceftazidime/avibactam between KPC and MBL isolates. Checkerboard analysis revealed appreciable synergy between combinations of tigecycline (35%) or eravacycline (40%) with polymyxin-B. The synergy rates for the combination of tigecycline or eravacycline with polymyxin-B were similar among the KPC and MBL isolates. These combinations maintained a synergy rate of 70.6% even against polymyxin-B resistant isolates. In contrast, combinations of tigecycline (5%) or eravacycline (10%) with ceftazidime/avibactam showed significantly lower synergy than combinations with polymyxin-B (P < 0.001 and P = 0.002, respectively). Among the MBL CRKP isolates, only one exhibited synergy with eravacycline or tigecycline and ceftazidime/avibactam combinations, and no synergistic activity was identified in the time-kill analysis for these combinations. The combination of eravacycline and polymyxin-B demonstrated the most promising synergy in the time-kill analysis. CONCLUSION: This study provides substantial evidence of a significant synergy when combining tigecycline or eravacycline with polymyxin-B against CRKP strains, including those producing MBL. These results highlight potential therapeutic strategies against CRKP infections.

Lipid metabolism-related long noncoding RNA RP11-817I4.1 promotes fatty acid synthesis and tumor progression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common types of tumors. The influence of lipid metabolism disruption on the development of HCC has been demonstrated in published studies. AIM: To establish an HCC prognostic model for lipid metabolism-related long non-coding RNAs (LMR-lncRNAs) and conduct in-depth research on the specific role of novel LMR-lncRNAs in HCC. METHODS: Correlation and differential expression analyses of The Cancer Genome Atlas data were used to identify differentially expressed LMR-lncRNAs. Quantitative real-time polymerase chain reaction analysis was used to evaluate the expression of LMR-lncRNAs. Nile red staining was employed to observe intracellular lipid levels. The interaction between RP11-817I4.1, miR-3120-3p, and ATP citrate lyase (ACLY) was validated through the performance of dual-luciferase reporter gene and RIP assays. RESULTS: Three LMR-lncRNAs (negative regulator of antiviral response, RNA transmembrane and coiled-coil domain family 1 antisense RNA 1, and RP11-817I4.1) were identified as predictive markers for HCC patients and were utilized in the construction of risk models. Additionally, proliferation, migration, and invasion were reduced by RP11-817I4.1 knockdown. An increase in lipid levels in HCC cells was significantly induced by RP11-817I4.1 through the miR-3120-3p/ACLY axis. CONCLUSION: LMR-lncRNAs have the capacity to predict the clinical characteristics and prognoses of HCC patients, and the discovery of a novel LMR-lncRNAs, RP11-817I4.1, revealed its role in promoting lipid accumulation, thereby accelerating the onset and progression of HCC.

Marginal zone lymphoma with severe rashes: A case report.

BACKGROUND: Marginal zone lymphoma (MZL) is an indolent subtype of non-Hodgkin lymphoma (NHL), which is rare clinically with severe rashes as the initial symptom. CASE SUMMARY: This study reports a case of MZL with generalized skin rashes accompanied by pruritus and purulent discharge. First-line treatment with rituximab combined with zanubrutinib had poor effects. However, after switching to obinutuzumab combined with zanubrutinib, the case was alleviated, and the rashes disappeared. CONCLUSION: For patients with advanced stage MZL not benefiting from type I anti-CD20 monoclonal antibody (mAb) combination therapy, switching to a type II anti-CD20 mAb combination regimen may be considered. This approach may provide a new perspective in the treatment of MZL.

Risk factors for mortality after linezolid treatment of vancomycin-resistant Enterococcus bloodstream infection.

OBJECTIVES: We analyzed the risk factors affecting linezolid treatment outcome in vancomycin-resistant Enterococcus (VRE) bloodstream infection (BSI). METHODS: We conducted a multicenter observational study of patients who received linezolid 600 mg every 12 hours for VRE BSI. The primary outcome was 28-day mortality. The estimated area under the concentration-time curve and trough concentration were calculated. Multivariable logistic regression was used for the outcome analysis. RESULTS: A total of 170 patients were included: 114 (67.1%) survived and 56 (32.9%) did not. A total of 26 (18.2%) isolates showed a linezolid minimum inhibitory concentration (MIC) of ≤1 mg/l, 113 (79.0%) of 2 mg/l, and 4 (2.8%) of 4 mg/l. The univariable analysis showed that the linezolid MIC and concentration-time curve/MIC were not associated with mortality (P = 0.95 and P = 0.42, respectively). After adjusting for underlying comorbidity and disease severity, the linezolid dose per body weight (LDBW), body height, and interaction between them were independent risks for mortality. Marginal analysis showed that increasing the LDBW was protective in patients with a body height <160 cm. A trough concentration of >12.2 mg/l was a risk factor for thrombocytopenia. CONCLUSION: The LDBW and body height were interactively associated with clinical outcomes of linezolid treatment for VRE BSI.

The Combination of Daptomycin with Fosfomycin is More Effective than Daptomycin Alone in Reducing Mortality of Vancomycin-Resistant Enterococcal Bloodstream Infections: A Retrospective, Comparative Cohort Study.

INTRODUCTION: High-dose daptomycin-based combinations are recommended for vancomycin-resistant Enterococcus (VRE) bloodstream infection (BSI). Preclinical data have shown a synergistic effect of daptomycin/fosfomycin combinations against VRE. However, clinical studies comparing daptomycin monotherapy with daptomycin/fosfomycin combinations are unavailable. METHODS: An observational study of VRE-BSI was performed between 2010-2021 on patients receiving daptomycin monotherapy (≥ 8 mg/kg) or daptomycin combined with intravenous fosfomycin. Patients treated with concomitant ß-lactam combinations were excluded. The primary outcome was in-hospital mortality. Outcomes were analyzed using multivariable logistic regression and augmented inverse probability weighting (AIPW) analyses. RESULTS: Among 224 patients, 176 received daptomycin monotherapy, and 48 received fosfomycin combinations. The median daptomycin and fosfomycin doses were 9.8 mg/kg and 12 g/day, respectively. In-hospital mortality was 77.3% and 47.9% in the daptomycin monotherapy and fosfomycin combination groups (P < 0.001), respectively. Multivariable logistic regression analysis predicted lower mortality with fosfomycin combination treatment (adjusted odds ratio, 0.35; 95% confidence interval (CI), 0.17-0.73; P = 0.005). AIPW demonstrated a 17.8% reduced mortality with fosfomycin combinations (95% CI, - 30.6- - 4.9%; P = 0.007). The survival benefit was significant, especially among patients with a lower Pitt bacteremia score or fosfomycin minimum inhibitory concentration (MIC) ≤ 64 mg/l. Fosfomycin combination resulted in higher hypernatremia (10.4% vs. 2.8%, P = 0.04) and hypokalemia (33.3% vs. 15.3%, P = 0.009) compared to daptomycin monotherapy. CONCLUSION: The combination of high-dose daptomycin with fosfomycin improved the survival rate of patients with VRE-BSI compared to daptomycin alone. The benefit of the combination was most pronounced for VRE with fosfomycin MIC ≤ 64 mg/l and for patients with a low Pitt bacteremia score.

High-level expression of functional Pfu DNA polymerase recombinant protein by mimicking the enhanced green fluorescence protein gene codon usage.

Pfu DNA polymerase is a vital enzyme in PCR-related experiments. However, it is not easy to achieve high-level expression and high purity through one-step purification. This paper illustrates the method to acquire the full-length open reading frame of Pfu DNA polymerase. Without altering its amino acids, we have modified the codon usage, based on that of the enhanced green fluorescence protein (eGFP), and named it rPfu. The synthesized rPfu gene has been subcloned into the pET28a plasmid and expressed in four Escherichia coli strains without the pLysS plasmid. Three strains have expressed a high level of soluble Pfu DNA polymerase. With the aid of Ni-NTA His•Bind® resin, we could obtain high purity (>95%) soluble recombinant protein. Compared with the commercial, proofreading DNA polymerase, rPfu's bioactivity was 12,987 U/mg; that is, 88,311 U of rPfu could be obtained from 50 mL cultured E. coli. The purified rPfu was able to amplify the length of DNA fragments at least 5.5 kb. The method of increasing soluble protein's yield using the eGFP codon usage may introduce a new possibility to the expression of other soluble recombinant proteins.

The combination of daptomycin with ß-lactam antibiotics is more effective than daptomycin alone for vancomycin-resistant Enterococcus faecium bloodstream infection.

BACKGROUND: The treatment options for vancomycin-resistant Enterococcus (VRE) are limited. A combination of daptomycin (DAP) and ß-lactam (BL) has been suggested; however clinical studies supporting this are lacking. METHODS: Patients with VR E. faecium bacteremia who received ≥ 8 mg/kg daptomycin for ≥ 72 h and initiated ≤ 5 days of culture collection between 2010 and 2021 were included. DAP+BL was defined as receiving BL for ≥ 24 h and within 24 h of DAP initiation. The primary endpoint was a composite clinical success (neither 14-day mortality, microbiological failure, nor change in the anti-VRE regimen). Outcomes were analyzed using multivariable logistic regression and augmented inverse probability weighting (AIPW). RESULTS: A total of 430 patients were enrolled (DAP, n = 45; DAP+BL, n = 385). Clinical success was achieved in 19 (42.2%) patients in the DAP group and 244 (63.4%) in the DAP+BL group [adjusted odds ratio, 3.19; 95% confidence interval (CI) 1.61-6.33; P = 0.001]. Marginal analysis showed that the efficacy of DAP+BL was particularly significant with DAP dose ≥ 9 mg/kg and DAP minimum inhibitory concentration (MIC) ≥ 2 mg/L. With the balance of AIPW, standardized mean clinical success rates for DAP and DAP+BL 37.3% and 63.5%, respectively. The difference between DAP+BL and DAP was of 26.2% in favor of DAP+BL (95% CI, 10.0-42.3%; P = 0.001). CONCLUSIONS: DAP+BL was associated with a significantly higher rate of compositive clinical success than DAP for treatment of VR E. faecium bacteremia. The study suggested BL in combination with high-dose DAP for VR E. faecium bacteremia treatment, especially when VRE showed a high DAP MIC.

A High Daptomycin Dose Is Associated with Better Bacterial Clearance in Infections Caused by Vancomycin-Resistant Enterococcus faecium Regardless of Daptomycin Minimum Inhibitory Concentration in a Rat Infective Endocarditis Model.

A high daptomycin dose has been suggested for treating vancomycin-resistant Enterococcus faecium (VREf) infections. However, even a 12 mg/kg daptomycin dose might be insufficient for treating VREf with high daptomycin minimum inhibitory concentrations (MICs). Additionally, animal pharmacodynamic and infection models to confirm the efficacy of 12 mg/kg daptomycin are lacking. Male Wistar rats were used for pharmacokinetic profiling and for the development of an infective endocarditis (IE) model. Daptomycin-susceptible dose-dependent VREf (DSE) (MIC of 0.5 mg/L) and daptomycin nonsusceptible VREf (DNSE) (MIC of 8 mg/L) were used for the IE models. The bacterial load of vegetation was the primary outcome and was evaluated after 3 days of daptomycin treatment. Daptomycin administered subcutaneously (s.c.) at 45 and 90 mg/kg, which corresponded to maximum serum concentrations (Cmax) of 122.6 mg/L and 178.5 mg/L, respectively, was equivalent to doses of 8 mg/kg and 12 mg/kg, respectively, in humans. The Cmax/MIC value was correlated with the bacterial load of vegetation after treatment (r = -0.88, P < 0.001). The 90 mg/kg s.c. group showed a significantly lower bacterial load of vegetation (log10 CFU/g) than the 45 mg/kg s.c. group against DSE (0 versus 4.75, P < 0.001) and DNSE (5.12 versus 6.98, P = 0.002). The 90 mg/kg s.c. group did not sterilize the vegetation against DNSE. Although the human equivalent dose of 12 mg/kg daptomycin was more effective than the smaller dose in reducing the bacterial load in DSE and DNSE IE, the dose could not sterilize the vegetation during a DNSE treatment. Further treatment strategies by which to manage severe VREf infections, especially at high daptomycin MICs, are urgently needed. IMPORTANCE Using a rat IE model with pharmacokinetic analysis, the treatment response of VREf IE was found to be daptomycin dose-dependent, presented as Cmax/MIC or as the 24 h area under the concentration-time curve (AUC0-24)/MIC. Daptomycin 90 mg/kg s.c. significantly reduced the bacterial load against DSE and DNSE. It also showed significant activity against DSE and DNSE, compared to 45 mg/kg s.c. Although daptomycin 90 mg/kg can eradicate the bacterial load after 3 days of treatment against DSE, eradication cannot be achieved with 90 mg/kg daptomycin against DNSE.

Daptomycin area under the curve to minimum inhibitory concentration ratio by broth microdilution for predicting the outcome of vancomycin-resistant Enterococcus bloodstream infection.

OBJECTIVES: Different methods are used to determine the minimum inhibitory concentration (MIC) for daptomycin. The threshold is unknown for the free drug area under the concentration-time curve to MIC ratio (fAUC/MIC) of daptomycin using broth microdilution (BMD) to predict outcome of vancomycin-resistant enterococcus (VRE) bacteremia. The MIC testing method which is best for predicting the outcome remains unclear. METHODS: This is a retrospective cohort study. The inclusion criterion was VRE bacteremia treated with ≥ 8 mg/kg of daptomycin. As we aimed to compare different daptomycin MIC testing methods for predicting the clinical outcome of VRE bacteremia, the inclusion criteria included the availability of MIC values for BMD, Etest, and automated antimicrobial susceptibility testing (AST). The primary end point was 28-day mortality. The fAUC/MIC was dichotomized using classification and regression tree analysis for predicting survival. RESULTS: A total of 393 patients were included; 215 survived and 178 died. In the multivariable logistic model for predicting mortality, the dichotomized fAUC/MICs for Etest and AST were 0.508 and 0.065 times as probable, respectively, as that for BMD to minimize information loss. An fAUC/MIC > 75.07 for BMD significantly predicted lower mortality (adjusted odds ratio, 0.53, 95% confidence interval, 0.30-0.95; P = 0.03) after adjusting for underlying disease and bacteremia severity. Using Monte Carlo simulation, none of the doses had a probability of target attainment of ≥ 50% with an MIC of ≥ 2 mg/L. CONCLUSION: The dichotomized threshold for fAUC/MIC for BMD was the best predictor of mortality. An fAUC/MIC > 75.07 for BMD independently predicted better survival.

Minocycline Susceptibility and tetB Gene in Carbapenem-Resistant Acinetobacter baumannii in Taiwan.

Purpose: In this study, we evaluated the minocycline susceptibility rate in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical strains, and the association between tetB carriage and minocycline susceptibility in CRAB. Patients and Methods: A total of 100 genetically unrelated CRAB clinical strains from bloodstream infection were randomly collected from a medical center in Taiwan. An argument for a new minocycline susceptibility breakpoint of 1 mg/L was suggested based on pharmacokinetic (PK) and pharmacodynamic (PD) studies. Strains with minocycline minimum inhibitory concentrations (MICs) of >1 mg/L were classified as PK-PD non-susceptible. TetB carriage was detected by polymerase chain reaction (PCR). Results: Fifty-five (55%) CRAB strains were susceptible to minocycline according to the Clinical and Laboratory Standards Institute (CLSI) criteria, among which 98.2% (54/55) were PK-PD non-susceptible. The minocycline MIC50/90 was 4/16 mg/L. Ninety-seven (97%) strains carried tetB. All of the tetB-positive strains and 66.7% (2/3) of the tetB-negative strains were PK-PD non-susceptible. By statistical analysis, tetB carriage was significantly correlated with PK-PD non-susceptibility (P = 0.03) and a higher minocycline MIC (P = 0.02). The sensitivity and specificity of the tetB PCR for predicting PK-PD non-susceptibility were 98% and 100%, respectively. Conclusion: At our institute, most CRAB strains were PK-PD non-susceptible and most carried tetB gene. Recognizing the minocycline MIC and tetB status may be essential when using minocycline to treat CRAB-related infections.

Influence of daptomycin doses on the outcomes of VRE bloodstream infection treated with high-dose daptomycin.

OBJECTIVES: The CLSI recommended high-dose daptomycin (8-12 mg/kg) for treating Enterococcus faecium bloodstream infections (BSI). The current study was designed to determine the safety and efficacy of increasing the daptomycin dose for VRE BSI patients receiving ≥8 mg/kg. METHODS: We conducted a multicentre prospective observational study of patients who received a ≥8 mg/kg dose of daptomycin for treatment of VRE BSI. The primary outcome was 28 day mortality. RESULTS: A total of 661 patients were included. The 28 day mortality rate was 45.1%. The survivors received higher doses of daptomycin than non-survivors (10.1 versus 9.8 mg/kg; P < 0.001). An increase in the daptomycin dose independently predicted lower mortality [adjusted OR (aOR) = 0.85; 95% CI = 0.73-0.99; P = 0.03]. Eighty-six survivors (23.7%) and 43 non-survivors (14.4%) received a ≥11 mg/kg dose of daptomycin (P = 0.003). The 8 to <11 and ≥11 mg/kg doses of daptomycin differed in the 28 day mortality in the higher MIC group (≥2 mg/L) (49.4% versus 33.3%; P = 0.004), but not in the lower MIC group (≤1 mg/L) (29.3% versus 29.4%; P = 0.99). A dose of ≥11 mg/kg was associated with a higher (3.9%) rate of highly elevated creatine kinase (>2000 U/L) compared with 1.1% with 8 to <11 mg/kg (P = 0.04). CONCLUSIONS: The efficacy of daptomycin is dose dependent. A high daptomycin dose, especially at ≥11 mg/kg, improved survival in patients with VRE BSI, but was associated with highly elevated creatine kinase. We recommend a ≥11 mg/kg dose of daptomycin be considered for treatment of VRE BSI, particularly for isolates with higher MICs.

Association of capsular polysaccharide locus 2 with prognosis of Acinetobacter baumannii bacteraemia.

Acinetobacter baumannii causes healthcare-associated infections worldwide. Capsular polysaccharide (CPS) is shown an important virulence factor of A. baumannii both in vitro and in vivo. Capsule locus 2 (KL2) for CPS is the most common KL type and is associated with carbapenem resistance. It is unclear whether KL2 is related to the clinical outcome of invasive A. baumannii infection. Here we had followed patients with A. baumannii bacteraemia prospectively between 2009 and 2014. One-third of the unduplicated blood isolates were randomly selected each year for microbiological and clinical studies. The KL2 gene cluster was identified using polymerase chain reaction. A total of 148 patients were enrolled randomly. Eighteen isolates (12.2%) carried KL2, and 130 isolates (87.8%) didn't. Compared with non-KL2 isolates, KL2 isolates had significantly higher resistance to imipenem, sulbactam, and tigecycline. Compared with the non-KL group, in the KL2 group, the hospital stay before development of bacteraemia was longer (P < 0.001), a higher percentage had pneumonia (P = 0.004), and the white blood cell count was lower (P = 0.03). Infection with KL2 A. baumannii predicted mortality (adjusted hazard ratio [aHR], 2.03; 95% confidence interval [CI], 1.09-3.78; P = 0.03), independently of the Pitt bacteraemia score (aHR, 1.34; 95% CI, 1.23-1.46; P < 0.001) and leucopenia (aHR, 2.16; 95% CI, 1.30-3.57; P = 0.003). Thrombocytopenia contributed to the effect of KL2 on mortality in bacteraemia (Sobel test P = 0.01). Large-scale studies are warranted to confirm these findings and the underlying mechanisms deserve further investigation.

Identification of Prognostic Biomarkers Originating From the Tumor Stroma of Betel Quid-Associated Oral Cancer Tissues.

BACKGROUND: Partial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging. METHODS: Gene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan-Meier curves of estimated overall survival were compared for statistical difference using the log-rank test. RESULTS: Compared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types. CONCLUSIONS: As the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.

The Tip Region on VP2 Protein of Bluetongue Virus Contains Potential IL-4-Inducing Amino Acid Peptide Segments.

Bluetongue is an infectious viral hemorrhagic disease of domestic and wild ruminants that has a considerable economic impact on domestic ruminants. There are currently at least 29 serotypes of bluetongue virus (BTV) in the world. Noteworthily, the pathogenesis among BTV serotypes is different, even in the same animal species. In this study, BTV2/KM/2003 and BTV12/PT/2003 were used to investigate the differential immunological effects on bovine peripheral blood mononuclear cells (PBMCs). The BTV viral load and the expression of cytokine messenger RNA (mRNA) in PBMCs were measured by fluorescence-based real-time reverse-transcription PCR (qRT-PCR). The immunofluorescence assay (IFA) was applied to detect BTV signals in monocyte-derived macrophages (MDMs). The SWISS-MODEL and IL-4pred prediction tools were used to predict the interleukin 4 (IL-4)-inducing peptides in BTV-coat protein VP2. Synthetic peptides of VP2 were used to stimulate PBMCs for IL-4-inducing capability. This study demonstrated that the cytokine profiles of BTV-induced PBMCs were significantly different between BTV2/KM/2003 and BTV12/PT/2003. BTV2 preferentially activated the T helper 2 (Th2) pathway, represented by the early induction of IL-4, and likely fed back to inhibit the innate immunity. In contrast, BTV12 preferentially activated the innate immunity, represented by the induction of tumor necrosis factor -α (TNF-α) and interleukin 1 (IL-1), with only minimal subsequent IL-4. The BTV nonstructural protein 3 antibody (anti-BTV-NS3) fluorescent signals demonstrated that monocytes in PBMCs and MDMs were the preferred targets of BTV replication. Bioinformatics analysis revealed that the capability to induce IL-4 was attributed to the tip region of the VP2 protein, wherein a higher number of predicted peptide segments on BTVs were positively correlated with the allergic reaction reported in cattle. Synthetic peptides of BTV2-VP2 induced significant IL-4 within 12-24 h post-infection (hpi) in PBMCs, whereas those of BTV12 did not, consistent with the bioinformatics prediction. Bovine PBMCs and synthetic peptides together seem to serve as a good model for pursuing the BTV-induced IL-4 activity that precedes the development of an allergic reaction, although further optimization of the protocol is warranted.

Type I hypersensitivity is induced in cattle PBMC during Bluetongue virus Taiwan isolate infection.

Bluetongue is a fatal viral disease in ruminants and has serious economic impacts on the livestock industry. Interactions between bluetongue virus (BTV) and immune cells are interesting because of the unique scenarios in each combination of animal species/breed and viral virulence/serotype. This study investigated the immune response in bovine peripheral blood mononuclear cells (PBMC) infected by the BTV2 Taiwan strain. The replication of the virus was limited in monocytes and monocyte-derived macrophages (MDM), and lymphocytes were less permissive. The cytokine mRNA of IL-4 in PBMC was expressed earlier and in greater quantities than that of innate immunity (TNFα, IL-1ß) and cell mediated immunity (CMI) (IFNγ), and the IL-4 protein was stably present in the culture medium until 72 h post-infection (hpi). Even in MDM reconstituted with autologous lymphocyte (MDM-Lymphocyte), the IL-4 still had high mRNA expression level. The level of IgE antibody also increased at 24-72 hpi, suggestive of the engagement of type I hypersensitivity in the pathogenesis. The anti-viral activity contained in the culture supernatant was transferrable to recipient infected PBMC from other cows. However, in infected MDM largely free of lymphocytes, mRNA expressions of IL-1ß, TNFα and IL-12p40 were normally expressed from 6 to 48 hpi, supporting the notion that IL-4 elaborated by lymphocytes in PBMC mediated the inhibition of both innate immunity and CMI to BTV2. The sum of responses subsequent to the early IL-4 expression likely constitutes part of the unique scenario in the current BTV2-Cow experimental combination biased toward Th2 response.

Heparan sulfate targeting strategy for enhancing liposomal drug accumulation and facilitating deep distribution in tumors.

Nanoparticles (NPs), such as liposomes, effectively evade the severe toxicity of unexpected accumulation and passively shuttle drugs into tumor tissues by enhanced permeability and retention. In the case of non-small cell lung cancer and pancreatic ductal adenocarcinoma, cancer-associated fibroblasts promote the aggregation of a gel-like extracellular matrix that forms a physical barrier in the desmoplastic stroma of the tumor. These stroma are composed of protein networks and glycosaminoglycans (GAGs) that greatly compromise tumor-penetrating performance, leading to insufficient extravasation and tissue penetration of NPs. Moreover, the presence of heparan sulfate (HS) and related proteoglycans on the cell surface and tumor extracellular matrix may serve as molecular targets for NP-mediated drug delivery. Here, a GAG-binding peptide (GBP) with high affinity for HS and high cell-penetrating activity was used to develop an HS-targeting delivery system. Specifically, liposomal doxorubicin (L-DOX) was modified by post-insertion with the GBP. We show that the in vitro uptake of L-DOX in A549 lung adenocarcinoma cells increased by GBP modification. Cellular uptake of GBP-modified L-DOX (L-DOX-GBP) was diminished in the presence of extracellular HS but not in the presence of other GAGs, indicating that the interaction with HS is critical for the cell surface binding of L-DOX-GBP. The cytotoxicity of doxorubicin positively correlated with the molecular composition of GBP. Moreover, GBP modification improved the in vivo distribution and anticancer efficiency of L-DOX, with enhanced desmoplastic targeting and extensive distribution. Taken together, GBP modification may greatly improve the tissue distribution and delivery efficiency of NPs against HS-abundant desmoplastic stroma-associated neoplasm.

Multicenter Study of Azole-Resistant Aspergillus fumigatus Clinical Isolates, Taiwan1.

In a multicenter study, we determined a prevalence rate of 4% for azole-resistant Aspergillus fumigatus in Taiwan. Resistance emerged mainly from the environment (TR34/L98H, TR34/L98H/S297T/F495I, and TR46/Y121F/T289A mutations) but occasionally during azole treatment. A high mortality rate observed for azole-resistant aspergillosis necessitates diagnostic stewardship in healthcare and antifungal stewardship in the environment.

Alpinia oxyphylla Fruit Extract Ameliorates Experimental Autoimmune Encephalomyelitis through the Regulation of Th1/Th17 Cells.

Alpinia oxyphylla is a traditional Chinese medicine widely used for treating diarrhea, ulceration, and enuresis. Moreover, A. oxyphylla is effective for cognitive function improvement and nerve regeneration. Multiple sclerosis (MS) is a chronic neuronal inflammatory autoimmune disease that commonly affects young adults in high-latitude regions. The aim of this study was to evaluate the beneficial effects of A. oxyphylla in an experimental autoimmune encephalomyelitis (EAE) mouse model, which is an extensively used model for human MS. The ethanolic extract of A. oxyphylla fruit (AO-1) was orally administered to EAE mice. Our results showed AO-1 significantly reduced EAE symptoms. Histopathological analysis showed AO-1 reduced demyelination, inflammation, gliosis, and axonal swelling in the spinal cord. Furthermore, immunohistochemistry and quantitative polymerase chain reaction (qPCR) studies revealed that the infiltration of CD4+, CD8+ T cells, and CD11b+ monocytes into the spinal cord decreased in the AO-1-treated group. Mechanistically, the Th1 transcription factor T-bet, Th17 transcription factor retinoic acid receptor-related orphan receptor γ (RORγt), and inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17 were reduced in the spinal cords of mice treated with AO-1. The expression levels of T-bet and RORγt were also lowered in the spleens of those mice. Further in vitro study showed AO-1 inhibited production of IFN-γ, IL-2, and tumor necrosis factor-α from MOG35-55-peptide-stimulated splenocytes. One component isolated from AO-1, yakuchinone A, inhibited IL-17 production in vitro and reduced EAE symptoms in the mice. Collectively, our results indicate that AO-1 ameliorated the severity of EAE in mice and may involve the regulation of Th1/Th17 response. A. oxyphylla warrants further investigation, particularly regarding its clinical benefits for MS.

Should we treat patients with only one set of positive blood cultures for extensively drug-resistant Acinetobacter baumannii the same as multiple sets?

Acinetobacter species are not considered skin commensals and under-treatment is an overriding concern when caring for critically-ill patients who are mostly at risk of extensively drug-resistant Acinetobacter baumannii (XDRAB) infections. Hence even a single blood culture yielding XDRAB will tend to prompt intervention. However, field observations suggest that patients with single-positive blood cultures had milder disease and were more likely to be recruited in interventional studies than those with multiple-positive blood cultures, yet no distinction is made in current clinical or trial recruitment practices. To our knowledge, this is the first study to compare the clinical characteristics and outcomes of patients with single-positive versus multiple-positive blood cultures for XDRAB. In this multicenter prospective cohort study of XDRAB bacteremic patients from July 2010 to June 2015, only patients with at least two simultaneously drawn blood cultures were included. The patients were classified as having single-positive or multiple-positive blood cultures according to the number of positive blood cultures yielding XDRAB. The primary end-point was the 28-day mortality. Of a total of 155 patients enrolled, 69 had a single-positive and 86 had multiple-positive blood cultures. Leukopenia (37.2% vs. 16.2%; P = 0.004), thrombocytopenia (56.0% vs. 26.5%; P < 0.001), higher Pitt bacteremia scores (6.6 vs. 5.5, P = 0.03) and higher 28-day mortality rates (70.9% vs. 43.5%; P = 0.001) distinguished patients with multiple-positive from those with single-positive cultures. Multivariate logistic regression showed that multi-positivity independently predicted 28-day mortality (adjusted odds ratio, 2.34; 95% confidence interval (CI), 1.03-5.28; P = 0.04) and the Cox regression confirmed that multi-positivity (adjusted hazard ratio, 1.80; 95% CI, 1.13-2.85; P = 0.01) predicted rapid mortality. Patients with multiple versus single positive blood cultures yielding XDRAB had greater morbidity and mortality. Investigators and clinicians should be aware that the blood culture positivity rate impacts outcomes of XDRAB bacteremia.

The urinary shedding of porcine teschovirus in endemic field situations.

Porcine teschoviruses (PTVs) belong to the genus Teschovirus within the family Picornaviridae. PTVs are universal contaminants in pig herds in endemic and multi-infection statuses. Previous research has demonstrated PTV antigens and nucleic acid in renal glomeruli and tubular epithelia, suggesting the possibility that PTVs might be shed and transmitted via urine. The study aimed to demonstrate, in the context of pathogenesis, the presence of PTVs in the urine of naturally infected pigs. Viral loads of fluid and tissue samples quantified by an established qRT-PCR showed detection rates of 100% by head and in urine, feces, plasma and nasal swabs, and 38% in kidney. As predicted, PTVs were present in urine at 10(4.02 ± 1.45) copies/100 µl volume, equivalent to 17% of that in plasma. No significant differences were observed between healthy and culled pigs or among the 7 sampled herds. The presence of PTVs in urine was further substantiated by molecular serotyping. In particular, PTV-10 was identified in the urine of 3 piglets from 3 separate herds, consistent with the most prevalent serotype found in this study, and in plasma. The urine mixes with feces to form slurry making it easier for PTV to spread and contaminate the environment.