A Polymeric Nanoparticle Formulation for Targeted mRNA Delivery to Fibroblasts.

Messenger RNA (mRNA)-based therapies offer enhanced control over the production of therapeutic proteins for many diseases. Their clinical implementation warrants formulations capable of delivering them safely and effectively to target sites. Owing to their chemical versatility, polymeric nanoparticles can be designed by combinatorial synthesis of different ionizable, cationic, and aromatic moieties to modulate cell targeting, using inexpensive formulation steps. Herein, 152 formulations are evaluated by high-throughput screening using a reporter fibroblast model sensitive to functional delivery of mRNA encoding Cre recombinase. Using in vitro and in vivo models, a polymeric nanoformulation based on the combination of 3 specific monomers is identified to transfect fibroblasts much more effectively than other cell types populating the skin, with superior performance than lipid-based transfection agents in the delivery of Cas9 mRNA and guide RNA. This tropism can be explained by receptor-mediated endocytosis, involving CD26 and FAP, which are overexpressed in profibrotic fibroblasts. Structure-activity analysis reveals that efficient mRNA delivery required the combination of high buffering capacity and low mRNA binding affinity for rapid release upon endosomal escape. These results highlight the use of high-throughput screening to rapidly identify chemical features towards the design of highly efficient mRNA delivery systems targeting fibrotic diseases.

Occurrence and predictors of delirium in critically ill older patients: a prospective cohort study.

Objectives: This study aims to analyze the occurrence of delirium in critically ill older patients and to identify predictors of delirium. Methods: This prospective study included critically ill older patients admitted into level II units of Intensive Care Medicine Department of a University Hospital. Patients with Glasgow Coma Scale score ≤11, traumatic brain injury, terminal disease, history of psychosis, blindness/deafness, or inability to understanding/speaking Portuguese were excluded. The Confusion Assessment Method-Short Form (CAM-4) was used to assess the presence of delirium. Results: The final sample (n = 105) had a median age of 80 years, most being female (56.2%), widowed (49.5%), and with complete primary education (53%). Through CAM-4, 36.2% of the patients had delirium. The delirium group was more likely to have previous cognitive decline (48.6% vs 19.6%, P = .04) and severe dependency in instrumental activities of daily living (34.3% vs 14.8%, P = .032), comparing with patients without delirium. The final multiple logistic regression model explained that patients with previous cognitive decline presented a higher risk for delirium (odds ratio: 4.663, 95% confidence Interval: 1.055-20.599, P = .042). Conclusions: These findings corroborate previous studies, showing that cognitive decline is an independent predictor for delirium in older patients. This study is an important contribution for the knowledge regarding the predictors of delirium. The recognition of these factors will help to identify patients who are at high risk for this syndrome and implement early screening and prevention strategies. However, further studies with larger samples, recruited from other clinical settings as well as analyzing other potential factors for delirium, will be needed.

Incidence of hypertension in a prospective cohort study of adults from Porto, Portugal.

BACKGROUND: During the past 30 years, Portugal has been described as one of the countries with highest median blood pressure levels in Europe, but the incidence of hypertension is unknown. The aim of this study was to estimate the incidence of hypertension, according to socio-demographic characteristics and lifestyles. METHODS: A population-based cohort of randomly selected dwellers from Porto, Portugal, aged ≥ 18 years, was assembled in 1999-2003 (EPIPorto study) and 796 hypertension-free individuals (62.6% women) were reassessed after a median of 3.8 years. Hypertension was defined as blood pressure ≥ 140/90 mmHg and/or antihypertensive drug therapy. Incidence rate ratios (IRR) were estimated using Poisson regression. RESULTS: The overall incidence rate was 47.3 [95% confidence interval (95% CI): 40.5-55.5] per 1000 person-years. Among women, the incidence was 43.4 (35.6-53.1) and among men 52.7 (41.3-68.0) per 1000 person-years. The incidence was lower in women up to 60 years and much higher among women above 60 (110.0 vs. 64.4 per 1000 person-years among men, p for age-sex interaction = 0.032). Participants with higher education had a lower risk of becoming hypertensive (≥ 13 years vs. ≤ 4 years: RR = 0.70, 95% CI, 0.46-1.08, p for linear trend <0.001), independently of age and sex. Overweight and obesity were associated with a 1.67-fold and 2.44-fold increased risk of hypertension, respectively, independently of age, sex and education. CONCLUSIONS: In this urban Portuguese population the incidence rate of hypertension was high, with new cases occurring predominantly among older subjects, the less educated and those with overweight-obesity. Despite recent progresses in blood pressure related outcomes, the risk of hypertension remains higher in Portugal than in other developed countries.

Recall of delirium and related distress in elderly hospitalized patients: A prospective study.

Background: Delirium is a very common neuropsychiatric disorder in the elderly, with a significant physical and psychological burden. Much is still unknown about its psychological effects. This study aims to identify the proportion of patients who recall delirium and to analyze the distress caused by it. In addition, this study aims to analyze the association between delirium recall and related distress and global psychological distress regarding hospitalization. Methods: This is a prospective study with elderly hospitalized patients in level-2 units of intensive care medicine department of a university hospital. Exclusion criteria were a Glasgow Coma Scale total ≤11, brain injury, blindness, deafness, or inability to communicate. Delirium was daily assessed with the Confusion Assessment Method. Delirium recall and related distress in patients were measured using the Delirium Experience Questionnaire. Global psychological distress was assessed with the Kessler Psychological Distress Scale. Results: From 105 patients, 38 (36.2%) developed delirium. Most patients did not remember the delirium episode (64.7%). Among those who remembered (35.3%), most described delirium as a distressing experience (75%). Delirium recall was associated with high global psychological distress (P = .029). Conclusions: Distress related to delirium is high, namely in patients who recall the episode. Global psychological distress during hospitalization is associated with delirium recall. This study highlights the need to assess the experience of delirium in these patients, as well as the importance of providing support and psychological interventions to minimize the associated distress.

Nanoparticles for intracellular-targeted drug delivery.

Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

Population-based study on the prevalence of spirometric obstructive pattern in Porto, Portugal.

BACKGROUND: The diagnosis of obstructive lung disease (OLD) based on clinical grounds is challenging. There have been no population-based COPD studies that collected pulmonary function data in Portugal, a country in transition between phases 2 and 3 of the smoking epidemic. OBJECTIVE: To estimate the prevalence of obstructive pattern on spirometry in a representative sample of adults from Porto, Portugal. METHODS: We conducted a health survey between 2001 and 2003, and 758 participants ≥ 40 years old had reliable spirometry. We used a structured questionnaire to collect demographic, clinical, social, and behavioral data. Obstructive pattern was defined as FEV(1)/FVC < 70%. Logistic regression was performed to quantify the association between socio-demographic and clinical factors and outcome. RESULTS: The participants' mean ± SD age was 58.5 ± 11.5 years, and 62% were women. The prevalence of spirometric obstructive pattern was 10.7%, 95% CI 8.6-13.1%; 13.4% in men, and 9.1% in women (P = .08). The age-adjusted odds ratios for cumulative smoking exposure of less than and more than 20 pack years, in comparison with never smokers, were 3.49 (95% CI 1.02-11.92) and 3.91 (95% CI 1.29-11.89) among men, and 1.47 (95% CI 0.53-4.08) and 2.68 (95% CI 1.07-6.68) among women, respectively. Previously diagnosed OLD was reported by 30.9% (95% CI 21.1-42.1%) of the participants with spirometric obstructive pattern. Spirometry confirmed the OLD diagnosis in 20.5% (95% CI 13.7-28.7%) of subjects who self-reported OLD (kappa = 0.14, 95% CI 0.07-0.20). CONCLUSIONS: The prevalence of spirometric obstructive pattern was high. Considering Portugal's position in the smoking epidemic, together with the aging of the population, we can expect an increase in the prevalence of OLD in older people and in women. Our results confirm the limited validity of self-reported OLD in epidemiological studies.

B-type natriuretic peptide is related to cardiac function and prognosis in hospitalized patients with decompensated cirrhosis.

BACKGROUND: B-type natriuretic peptide (BNP) concentrations are high in cirrhosis, possibly related to volume status and cirrhotic cardiomyopathy. The prognostic significance of BNP in cirrhosis is unknown. AIMS: We aimed to evaluate (i) the influence of haemodynamic parameters and volaemia, assessed by impedance cardiography (ICG), in BNP levels, (ii) the performance of BNP as a prognostic marker, in a cohort of cirrhotic patients. METHODS: Patients consecutively hospitalized with decompensated cirrhosis during 1 year were evaluated. At admission, ICG and BNP measurements were performed in 83 patients (median age 56 years; median Child-Pugh score=10). The 70 patients discharged were followed for the occurrence of death within 6 months. RESULTS: Median BNP levels were 130.3 (65.2-363.3) pg/ml. Independent BNP predictors in multivariate linear regression analysis were cardiac output, age and haemoglobin (R(2)=36.7%). The 24 patients with cardiac systolic dysfunction, defined by low cardiac output, had higher BNP concentrations than the other patients (230.8 vs 98.5 pg/ml, P=0.003). BNP levels above median were associated with an increased occurrence of death within 6 months of discharge (log rank P=0.023). Cardiac output and BNP were predictors of survival in univariate Cox regression analysis. Only BNP remained independently related to the outcome in multivariate analysis [hazard ratio=2.86 (1.11-7.38), P=0.03]. CONCLUSIONS: BNP levels in cirrhosis reflect cardiac systolic function and non-cardiac variables that should be considered in their interpretation. BNP is an independent predictor of medium-term survival in advanced cirrhosis, suggesting its utility in risk stratification of decompensated cirrhotic patients.

Antifungal nanoparticles and surfaces.

Nosocomial fungal infections, an increasing healthcare concern worldwide, are often associated with medical devices. We have developed antifungal nanoparticle conjugates that can act in suspension or attach to a surface, efficiently killing fungi. For that purpose, we immobilized covalently amphotericin B (AmB), a potent antifungal agent approved by the FDA, widely used in clinical practice and effective against a large spectrum of fungi, into silica nanoparticles. These antifungal nanoparticle conjugates are fungicidal against several strains of Candida sp., mainly by contact. In addition, they can be reused up to 5 cycles without losing their activity. Our results show that the antifungal nanoparticle conjugates are more fungistatic and fungicidal than 10 nm colloidal silver. The antifungal activity of the antifungal nanoparticle conjugates is maintained when they are immobilized on a surface using a chemical adhesive formed by polydopamine. The antifungal nanocoatings have no hemolytic or cytotoxic effect against red blood cells and blood mononuclear cells, respectively. Surfaces coated with these antifungal nanoparticle conjugates can be very useful to render medical devices with antifungal properties.

A Fluorescence-Based Sensor Assay that Monitors General Protein Aggregation in Human Cells.

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aß-peptide. Exposure to proteome destabilizers lead to re-localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide-genetic screens to identify genes and pathways involved in protein misfolding and aggregation.

Endocarditis, Meningitis and Pneumocystis Pneumonia.

Infective endocarditis (IE) is associated with high morbidity and mortality despite advances in antibiotic and surgical treatment. Systemic embolism occurs in up to 49% of IE patients and may involve the major arteries, limb arteries, viscera and the central nervous system. In this report we describe a 60-year-old female patient with a history of acute lymphoblastic leukaemia who presented with endocarditis manifesting as stroke, acute limb ischaemia and meningitis. Early diagnosis is essential since treatment lowers the risk of embolism, with most events occurring within 2 weeks of treatment initiation. LEARNING POINTS: Infective endocarditis has a very variable clinical presentation and should be suspected in patients with multisystemic involvement especially if vascular and neurological systems are affected.As early diagnosis is essential since treatment lowers the risk of embolism, echocardiography should be more widely available and possibly become a core medical skill.Because of the high mortality, a high index of suspicion and low threshold for investigation is essential in at-risk groups.

Leucobacter luti sp. nov., and Leucobacter alluvii sp. nov., two new species of the genus Leucobacter isolated under chromium stress.

Two strains designated RF6(T) and RB10(T) were isolated, from activated sludge and from river sediments, respectively, both systems receiving chromium contaminated water. Phylogenetic analysis showed that strain RF6(T)and strain RB10(T) represented two new species of the genus Leucobacter. Strain RB10(T) can be distinguished from RF6(T) by its ability to grow at 37 degrees C, by showing a different optimum pH, by cell wall amino acids different relative amount and by having the fatty acid strait C16:0 as the third most abundant fatty acid. On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain RF6(T) represents a new species of the genus Leucobacter for which we propose the name Leucobacter luti (CIP 108818(T)=LMG 23118) and that strain RB10(T) represents an additional new species of the same genus for which we propose the name Leucobacter alluvii (CIP 108819(T)=LMG 23117).

High Antimicrobial Activity and Low Human Cell Cytotoxicity of Core-Shell Magnetic Nanoparticles Functionalized with an Antimicrobial Peptide.

Superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with antimicrobial agents are promising infection-targeted therapeutic platforms when coupled with external magnetic stimuli. These antimicrobial nanoparticles (NPs) may offer advantages in fighting intracellular pathogens as well as biomaterial-associated infections. This requires the development of NPs with high antimicrobial activity without interfering with the biology of mammalian cells. Here, we report the preparation of biocompatible antimicrobial SPION@gold core-shell NPs based on covalent immobilization of the antimicrobial peptide (AMP) cecropin melittin (CM) (the conjugate is named AMP-NP). The minimal inhibitory concentration (MIC) of the AMP-NP for Escherichia coli was 0.4 µg/mL, 10-times lower than the MIC of soluble CM. The antimicrobial activity of CM depends on the length of the spacer between the CM and the NP. AMP-NPs are taken up by endothelial (between 60 and 170 pg of NPs per cell) and macrophage (between 18 and 36 pg of NPs per cell) cells and accumulate preferentially in endolysosomes. These NPs have no significant cytotoxic and pro-inflammatory activities for concentrations up to 200 µg/mL (at least 100 times higher than the MIC of soluble CM). Our results in membrane models suggest that the selectivity of AMP-NPs for bacteria and not eukaryotic membranes is due to their membrane compositions. The AMP-NPs developed here open new opportunities for infection-site targeting.

Differential internalization of amphotericin B--conjugated nanoparticles in human cells and the expression of heat shock protein 70.

Although a variety of nanoparticles (NPs) functionalized with amphotericin B, an antifungal agent widely used in the clinic, have been studied in the last years their cytotoxicity profile remains elusive. Here we show that human endothelial cells take up high amounts of silica nanoparticles (SNPs) conjugated with amphotericin B (AmB) (SNP-AmB) (65.4 ± 12.4 pg of Si per cell) through macropinocytosis while human fibroblasts internalize relatively low amounts (2.3 ± 0.4 pg of Si per cell) because of their low capacity for macropinocytosis. We further show that concentrations of SNP-AmB and SNP up to 400 µg/mL do not substantially affect fibroblasts. In contrast, endothelial cells are sensitive to low concentrations of NPs (above 10 µg/mL), in particular to SNP-AmB. This is because of their capacity to internalize high concentration of NPs and high sensitivity of their membrane to the effects of AmB. Low-moderate concentrations of SNP-AmB (up to 100 µg/mL) induce the production of reactive oxygen species (ROS), LDH release, high expression of pro-inflammatory cytokines and chemokines (IL-8, IL-6, G-CSF, CCL4, IL-1ß and CSF2) and high expression of heat shock proteins (HSPs) at gene and protein levels. High concentrations of SNP-AmB (above 100 µg/mL) disturb membrane integrity and kill rapidly human cells (60% after 5 h). This effect is higher in SNP-AmB than in SNP.

Antifungal activity of dental resins containing amphotericin B-conjugated nanoparticles.

OBJECTIVES: To evaluate the antifungal activity, biocompatibility and mechanical properties of dental resins containing silica nanoparticles functionalized with amphotericin B (SNP-DexOxAmB) against five species of Candida. METHODS: Dental resin composites (Spectrum, Dentsply DeTrey, GmbH, Germany) having 2% (w/w) of SNP-DexOxAmB (SNPs of 5 and 80nm, denoted as SNP5 and SNP80) were aged for 10, 20 and 30 days at 37°C, in phosphate buffer saline buffer pH 7.4 (PBS). At different time, the antifungal activity was evaluated by a direct contact assay against 1×10(4)cells of Candida. The biocompatibility of the resins was tested against human fibroblasts, endothelial cells and red blood cells. RESULTS: Dental resins containing SNP5-DexOxAmB have high (1×10(4)cells killed in 5h by ∼70mg of dental resin composite containing 2% (w/w) of SNP-DexOxAmB) and durable (for at least 1 month) antifungal activity against five strains of Candida. The incorporation of the nanoparticles (NPs) had no significant change in the mechanical properties of the resin, specifically the flexural strength and modulus. Our results further show that the antifungal activity is mainly mediated by direct contact and not by leaching of NPs from the resin. Resins incorporating SNP5-DexOxAmB have longer-term antifungal activity than SNP80-DexOxAmB. The antimicrobial activity of resins with SNP5-DexOxAmB persists after 4 cycles of re-use and it is superior to the activity obtained for dental resins containing silver NPs. In addition, dental resins incorporating SNP5-DexOxAmB are non-cytotoxic against human skin fibroblasts and human umbilical vein endothelial cells, and non-hemolytic against human red blood cells. SIGNIFICANCE: The incorporation of SNP5-DexOxAmB in dental resins resulted in a non-cytotoxic composite with high and durable antifungal activity.

Statin-induced low cholesterol is not associated with poor outcome in chronic heart failure.

BACKGROUND: Low cholesterol levels are associated with a worse outcome in patients with heart failure (HF). Use of statins in HF remains controversial. We aimed to assess whether the prognosis of patients with intrinsically low cholesterol levels differed from that of those with pharmacologically induced low cholesterol. MATERIALS AND METHODS: We conducted a retrospective cohort study on 464 ambulatory patients attending a specialized HF clinic. Patients were cross-classified according to statin therapy and admission total cholesterol level (low cholesterol <150 mg/dL and cholesterol ≥150 mg/dL): (1) low total cholesterol level on statin therapy; (2) low total cholesterol level not taking statins; (3) cholesterol ≥150 mg/dL on statin therapy; and (4) cholesterol ≥150 mg/dL not on statin therapy. Patients were followed up to 5 years and the outcome was all-cause death. A Cox regression analysis was used in prognosis assessment. RESULTS: Almost two thirds of the patients were men and the median population age was 69 years; 22.8% of the patients had preserved ejection fraction and 43.5% severe systolic dysfunction. The patients with an intrinsically low cholesterol had a hazard ratio of all-cause death up to 5 years of 2.38 (1.08-7.14) compared to those with low cholesterol induced by statin use. This association was independent of other variables associated with outcome. CONCLUSIONS: Patients with HF with instrisically low cholesterol levels have a double risk of death up to 5 years compared to patients with pharmacologically induced low cholesterol. Clinicians should not limit the use of statins by fear of lowering the cholesterol levels.

Higher C-reactive protein predicts worse prognosis in acute heart failure only in noninfected patients.

BACKGROUND: The prognostic role of C-reactive protein (CRP) in acute heart failure (HF) is not fully understood, and the impact of an infectious process in its risk-stratification power was not previously evaluated. HYPOTHESIS: As CRP is an inflammatory marker, its prognostic value in acute HF is probably different in patients with and without concurrent infection. METHODS: We recruited patients admitted to our hospital due to acute HF from October 2006 to October 2007. All patients were given treatment at the discretion of the attending physician. Serum CRP was measured at discharge in 225 patients. We followed patients for 3 months after discharge to assess occurrence of all-cause death or readmission due to HF. Infection was defined according to diagnoses registered on the discharge record. Patients were classified according to CRP tertiles, in the entire sample and in groups according to infection occurrence. RESULTS: : An infectious condition occurred in 109 patients (first and second CRP tertiles: 8.8 and 27.4 mg/L, respectively). No infection was detected in 116 patients (5.0 and 12.3 mg/L, respectively). In the group with infection, CRP was not a good predictor of adverse outcome. In the noninfected group, the hazard ratio of those with CRP > 12.3 mg/L was 2.46 (95% confidence interval: 1.29-4.70) in comparison with those with lower CRP. Adjusted hazard ratio for ischemic heart disease and diabetes was 2.03 (95% confidence interval: 1.06-3.91). CONCLUSIONS: CRP had no prognostic value in acute HF patients with an infectious complication. Noninfected patients with higher CRP at discharge had worse prognosis.

BNP at discharge in acute heart failure patients: is it all about volemia? A study using impedance cardiography to assess fluid and hemodynamic status.

BACKGROUND: Besides hemodynamic parameters, several other variables have been associated to B-type natriuretic peptide (BNP) levels. Limited knowledge on BNP determinants in acute heart failure (HF) can undermine the interpretation of BNP levels. METHODS AND RESULTS: To identify predictors of BNP levels, we evaluated 163 hospitalized acute HF patients. Thoracic fluid content (TFC) and hemodynamic parameters were measured by impedance cardiography at discharge. Patients were followed-up for 60 days for the occurrence of death/hospital admission. Median discharge BNP levels were 659.3 pg/ml. In multivariable linear regression analysis, TFC (ß=0.043, 95% CI 0.024-0.062 per U/kΩ, p<0.001) was a powerful predictor of BNP levels, independently of known markers of HF severity like severe systolic dysfunction and discharge New York Heart Association class. Other independent predictors were: new onset HF, albumin, and body mass index. Sex, left cardiac work index, stroke index, hemoglobin, renal failure and discharge furosemide and lisinopril doses were associated to BNP only in univariate analysis. During follow-up, 45 (27.6%) patients were hospitalized or died. TFC (HR=1.047 (1.016-1.080) per U/kΩ increase, p=0.003) and BNP (HR=1.003 (1.001-1.004) per 10 pg/ml increase, p<0.001) were univariate predictors of the outcome, but in multivariate Cox regression analysis, only BNP was independently associated with prognosis. CONCLUSION: Discharge BNP levels in acute HF patients reflected volemia and disease severity. Persistently high BNP levels during hospitalization should raise the possibility of remaining congestion, which could negatively influence prognosis. The utility of BNP as prognostic marker in HF may reside on its ability to reflect multiple underlying pathophysiological disturbances.