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This case emphasizes the importance of considering tuberculosis (TB) in the differential diagnosis of complex kidney imaging findings. We report a case of a 43-year-old male patient from India who presented with a five-day history of fever and vomiting. Laboratory results revealed metabolic acidosis, anemia, acute kidney injury, and elevated inflammatory markers. A computed tomography urogram demonstrated significant findings: minimal parenchymal remnants in the left kidney replaced by caliectasis, ureteral pyelitis, and retroperitoneal lymphadenopathy, including calcified nodes in the right kidney. The diagnosis of genitourinary TB was made after bronchoalveolar lavage and urine samples confirmed the presence of Mycobacterium tuberculosis . The imaging findings were consistent with "mastic kidney," a rare radiological sign of renal TB, characterized by extensive parenchymal destruction. First-line antituberculous therapy was initiated, leading to clinical improvement. This case highlights the critical role of early diagnosis and the importance of considering TB in patients with multisystem involvement. "Mastic kidney," though rare, remains a key radiological sign of renal TB, underscoring the need for heightened clinical awareness in suspected cases of extrapulmonary TB., Competing Interests: Human subjects: Consent for treatment and open access publication was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2025, Salvado de Morais et al.)

Introduction: Information about pan-genotypic treatments for hepatitis in Portugal is scarce. We aimed to evaluate the effectiveness and safety of glecaprevir plus pibrentasvir (GLE/PIB) treatment for hepatitis C virus (HCV) infection in real-world clinical practice., Methods: An observational prospective study was implemented in six hospitals with 121 adult HCV patients who initiated treatment with GLE/PIB between October 2018 and April 2019, according to clinical practice. Eligible patients had confirmed HCV infection genotype (GT) 1 to 6 and were either treatment-naïve or had experience with interferon-, ribavirin- or sofosbuvir-based regimens, with or without compensated cirrhosis. Baseline sociodemographic and safety data are described for the total population (N = 115). Effectiveness [sustained virologic response 12 weeks after treatment (SVR12)] and patient-reported outcomes are presented for the core population with sufficient follow-up data (n = 97)., Results: Most patients were male (83.5%), aged < 65 years (94.8%), with current or former alcohol consumption (77.3%), illicit drug use (72.6%), and HCV acquisition through intravenous drug use (62.0%). HIV co-infection occurred in 22.6% of patients. The prevalence of each GT was: GT1 51.3%, GT2 1.7%, GT3 30.4%, GT4 16.5%, and GT5.6 0%. Most patients were non-cirrhotic (80.9%) and treatment-naïve (93.8%). The SVR12 rates were 97.9% (95% CI: 92.8 - 99.4), and > 95% across cirrhosis status, GT, illicit drug use, alcohol consumption, and HCV treatment experience. The adverse event rate was 2.6%, and no patient discontinued treatment due to adverse events related to GLE/PIB., Conclusion: Consistent with other real-world studies and clinical trials, treatment with GLE/PIB showed high effectiveness and tolerability overall and in difficult-to-treat subgroups (ClinicalTrials.gov: NCT03303599).

An outbreak of hepatitis A is ongoing in Portugal, with 71 confirmed cases from 7 October 2023 to 24 April 2024. Most cases are male, aged 18-44 years, with many identifying as men who have sex with men (MSM) and reported as suspected sexual transmission. Phylogenetic analysis identified the subgenotype IA, VRD 521-2016 strain, last observed in an MSM-associated multi-country outbreak in 2016 to 2018. We wish to alert colleagues in other countries to investigate potential similar spread.

Introduction: Pseudomonas aeruginosa displays resistance to several available antibiotics. Infections caused by this pathogen are associated with a high mortality, morbidity, and considerable healthcare resource utilization and costs. This study was aimed at describing the use of ceftolozane/tazobactam (C/T) for the treatment of patients with P. aeruginosa infections., Methods: Case series analysis of hospitalized patients treated with C/T for P. aeruginosa infections in five public Portuguese hospitals. Patients presenting with infections caused by this pathogen and receiving C/T for at least 72h during hospitalization were eligible., Results: Sixty-four hospitalized patients with P. aeruginosa infections treated with C/T were evaluated between December 2016 and July 2019. Most patients were aged between 60 and 79 years (53.9%). Patients presented a total of 68 P. aeruginosa infections, with respiratory infections being the most common (28.1%, 18 out of 64). Most P. aeruginosa strains (85.9%, 55 out of 64) were extensively drug-resistant (XDR). C/T was mostly used as targeted therapy (98.4%, 63 out of 64 patients) and as monotherapy (72.7%, 47 out of 64 patients). Combination therapy was used in 47.4% (9 out of 19) of patients with bacteriemia. Most patients had successful microbiological (79.2%, 42 out of 53) and clinical (78.7%, 48 out of 61) outcomes. All-cause in-hospital mortality rate was 34.4%., Conclusion: The present case series contributes to the body of evidence suggesting that C/T is an effective and safe option for treating P. aeruginosa infections, namely those caused by XDR strains, both when used as mono- or combination therapy., (Copyright © 2022 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Integrase strand transfer inhibitor-based regimens are recommended for first-line therapy in human immunodeficiency virus type 2 (HIV-2). Nonetheless, dolutegravir (DTG) clinical trial data are lacking., Methods: We conducted a phase 2, single-arm, open-label trial to evaluate the safety and efficacy of a triple therapy regimen that included DTG in persons with HIV-2 (PWHIV-2) in Portugal. Treatment-naive adults receive DTG in combination with 2 nucleoside reverse transcriptase inhibitors (NRTIs). Treatment efficacy was evaluated by the proportion of patients who achieved a plasma viral load (pVL) <40 copies/mL and/or by the change from baseline in CD4+ T-cell count and in CD4/CD8 ratio at week 48., Results: A total of 30 patients were enrolled (22 women; median age, 55 years). At baseline, 17 (56.7%) individuals were viremic (median, pVL 190 copies/mL; interquartile range [IQR], 99-445). The median CD4 count was 438 cells/μL (IQR, 335-605), and the CD4/CD8 ratio was 0.8. Three patients discontinued the study. At week 48, all participants (27) had pVL <40 copies/mL. No virological failures were observed. Mean changes in CD4 count and CD4/CD8 ratio at week 48 were 95.59 cells/µL (95% confidence interval [CI], 28-163) and 0.32 (95% CI, .19 to .46). The most common drug-related adverse events were headache and nausea. One participant discontinued due to central nervous system symptoms. No serious adverse events were reported., Conclusions: DTG plus 2 NRTIs is safe and effective as first-line treatment for PWHIV-2 with a tolerability profile previously known. No virological failures were observed that suggest a high potency of DTG in HIV-2 as occurs in HIV-1., Clinical Trials Registration: M NCT03224338., Competing Interests: Potential conflicts of interest. A. R. S. reports consulting fees from and participation on a data and safety monitoring board (DSMB) or advisory board for MSD; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events; and support for attending meetings and/or travel from ViiV Healthcare, Gilead Sciences, and MSD. F. M. reports consulting fees from ViiV Healthcare, Gilead, Merck Sharp & Dohme, and Astra Zeneca; payment or honoraria for presentations from ViiV Healthcare, Gilead, and MercK Sharp & Dohme; and participation on an advisory board for ViiV Healthcare, Gilead, and MercK Sharp & Dohme. J. B. reports consulting fees from ViiV Healthcare and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Gilead and ViiV Healthcare. K. M. reports payment or honoraria as a conference speaker from Merck Sharp & Dome and ViiV Healthcare and payment for expert testimony for an advisory board from Gilead Sciences and ViiV Healthcare. N. M. reports payment or honoraria for speakers bureaus from Merck Sharpe & Dohme and Gilead Sciences. S. L. reports payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from ViiV Healthcare, MSD, Pfizer, and Gilead Sciences; support for attending meetings and/or travel from MSD; and participation on a DSMB or advisory board for ViiV Healthcare. D. T. reports payment or honoraria for presentations or educational events from ViiV Healthcare, Gilead, and MSD; support for attending an international meeting, scientific update from MSD; and participation on an advisory board from ViiV Healthcare, Gilead, and MSD. P. P. reports payment or honoraria for presentations from ViiV Healthcare, Gilead, MSD, and Janssen Cilag; support for attending meetings and/or travel from Janssen Cilag; and participation on an advisory board for Gilead Sciences, ViiV Healthcare, and MSD. P. G. reports grants or contracts from Gilead Sciences; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from ViiV Healthcare, Gilead Sciences, MSD, and Janssen; support for attending meetings and/or travel from ViiV Healthcare and MSD; and participation on a DSMB or advisory board from ViiV Healthcare and MSD. J. A. reports honoraria for an education event from ViiV Healthcare; support for attending meetings and/or travel from ViiV Healthcare; and participation on an advisory board from ViiV Healthcare. M. J. L. reports payment for a presentation from ViiV Healthcare. C. F. reports payment or honoraria as a specialist and speaker from ViiV Healthcare Unipessoal LDA and Gilead Sciences LDA and participation on an advisory board for ViiV Healthcare Unipessoal LDA and Gilead Sciences LDA. L. D. reports support for a virtual meeting registration from ViiV Healthcare; participation on an advisory board from Gilead Sciences; and a role as a member and paid collaborator from the GAT—Grupo de Ativistas em Tratamentos. D. P. reports consulting fees from ViiV Healthcare; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from ViiV Healthcare, AbbVie, and Roche; and support for attending meetings and/or travel from ViiV Healthcare and Merck Sharpe & Dohme. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A 50-year-old Caucasian man arrived at the emergency department presenting paucisymptomatic atrial fibrillation. Once discharged after the appropriate treatments, the patient continued to have paucisymptomatic episodes. For this reason, he was provided with the Cardionica device which made it possible to better investigate the type of arrhythmic episodes, in order to tailor his therapy and to finally restore a normal sinus rhythm in the patient.

Monkeypox, a neglected and re-emergent zoonotic disease caused by monkeypox virus (MPXV) infection, has been endemic in Central and Western Africa for decades. More recently, an outbreak has spread to a global level, occurring in sites with no previous reported cases and being clustered among men who have sex with men, suggesting new modes of transmission. There is an urgent need for research for a better understanding of the genomic evolution and changing epidemiology of the Orthopoxvirus group. Our work aimed to characterize the clinical and epidemiological features of a cohort of patients with MPXV infection in a Portuguese hospital, admitted between 5 May and 26 July 2022. In this retrospective observational study, aggregate data of a case series on the presentation, clinical course, and outcomes of confirmed MPXV infections are reported. The study included 40 men and 1 woman, with a mean age of 37.2 years old; 92.7% identified as men who have sex with men, 90.2% had unprotected sex or sex with multiple or anonymous partners in the previous month, and 39.0% reported to have had sex with an MPXV-confirmed case; 59.5% had previously known human immunodeficiency virus (HIV) infection, all of whom were under antiretroviral therapy, and no patients had acquired immunodeficiency syndrome (AIDS) criteria. About a quarter of patients were observed only a week after symptom onset. All patients had skin or mucosal lesions and the anogenital region was the most frequent lesion site. There were no statistically significant clinical differences between HIV-positive and negative individuals. Four patients were admitted to the inpatient clinic, two of whom had proctitis with difficult-to-manage anal pain. There were no reported deaths. Our findings suggest the sexual route as a relevant mode of transmission of MPXV and confirm the mostly benign presentation of this disease.

Introduction: Clostridioides difficile is the main cause of healthcare-associated diarrhea in Europe and North America. The aim of this study was to characterize the epidemiology and clinical burden of Clostridioides difficile infection among hospitalized patients in Portugal., Material and Methods: Retrospective study conducted in six public hospital centers in Portugal. All primary Clostridioides difficile infection episodes and related recurrences occurring in 2017, as well as episodes developing two to eight weeks after the last episode diagnosed in that year, were documented. The National Reference Laboratory (National Institute of Health Dr. Ricardo Jorge) provided national surveillance data on Clostridioides difficile infection., Results: A total of 385 inpatients with at least one primary episode diagnosed in 2017 were included. Most patients were aged over 70 years-old (73.2%). The included patients developed 451 episodes during the observation period. Approximately 44% of primary episodes were community-associated. Most episodes (94.9%) occurred in patients with one or more risk factors, with recent antibiotic exposure being particularly common (86.0%). All-cause in-hospital mortality was 19.5%, being significantly higher in patients aged over 65 years-old versus those aged 18 to 64 years-old (22.4% vs 7.8%, respectively). Over 50 different ribotypes were observed among 206 Clostridioides difficile strains received by the National Reference Laboratory., Conclusion: In Portugal, hospitalized patients with Clostridioides difficile infection are mostly older patients presenting risk factors for the development of this infection, particularly recent antibiotic exposure. Mortality is disproportionately high among the older population. Community-associated Clostridioides difficile infection is common among inpatients with this infection.

Competing Interests: Competing interests: We have read and understood BMJ policy on declaration of interests and have the following interests to declare. SL is employed by Sea4Us, a biotechnology company in marine pharmaceutical research. The other authors have nothing to declare.

Objective: Forceps delivery is associated with a higher risk of maternal birth trauma. It is speculated that it is due to sub-optimal use of forceps in inexperienced hands. The aim of this study was to determine the association between time of forceps birth and prevalence of levator avulsion over the last six decades., Study Design: This was a retrospective analysis of datasets of women with a history of forceps delivery, seen at a tertiary urogynaecological centre between January 2014 and August 2018. They had undergone a standardized interview, clinical examination and four-dimensional translabial ultrasound. Archived imaging data was reviewed for levator avulsion offline at a later date, blinded against all clinical data. Associations between levator avulsion, maternal age at first vaginal birth, the weight of the first vaginally born baby, and time since forceps delivery categorised by decade were tested by univariable analysis. Factors found to be significant on univariable analysis were included in a multivariable logistic regression model to test the association between prevalence of levator avulsion and time of forceps delivery while controlling for confounders., Results: In total, 2026 patients were seen during the study period. Among them 511 (25.2%) had a history of forceps delivery. Fourteen volume datasets were incomplete or missing, leaving 497 complete datasets for analysis. Mean age at presentation was 58 ± 12 years (23-91). Mean body mass index was 29 ± 6 kg/m 2 . Mean age at first delivery was 25 ± 5 years. Mean birth weight of the first vaginal birth was 3454 ± 557 g. 457 women (92%) had had one forceps delivery, 31 had two forceps deliveries (6%) and 9 had three forceps deliveries (2%). Mean time interval between forceps delivery and assessment was 32 ± 13 years (0.3-64.8). 229 women (46%) were diagnosed with levator avulsion. The prevalence of avulsion after forceps increased significantly from 34% to 56% between 1950 and 2017 (P = 0.04). However this difference became insignificant when controlling for maternal age at 1st vaginal delivery and birth weight., Conclusions: We found no evidence of a changed prevalence of levator avulsion at forceps delivery over the last 67 years., Competing Interests: Declaration of Competing Interest KL Shek and HP Dietz have received speaker’s fees and travel support from GE Medical and Mindray. HP Dietz has acted as consultant for GE Medical and Materna. The other authors have no conflict of interest to declare., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hepatitis E virus genotype 3 infections are normally asymptomatic in immunocompetent individuals. Symptomatic cases of acute icteric hepatitis E are seldom observed among women, younger men and children but are particularly seen in middle-aged/elderly men. We report a case of severe acute hepatitis E caused by genotype 3 in an immunocompetent 40-year-old woman that required prolonged hospitalization. Her medical history included an autoimmune background, namely atrophic gastritis and Graves' disease. She presented an extensive hepatic necrosis as revealed by the high levels of aminotransferases (ALT 4893 U/L; AST 3138 U/L). She showed also a coagulation disorder (prothrombin time; INR = 1.33). Serological markers for hepatitis viruses A, B and C were negative but serum was positive for hepatitis E virus RNA. Sequencing and phylogenetic analysis revealed that the hepatitis E virus strain belonged to subgenotype 3a. This is suggestive of an association between the severe acute hepatitis E virus genotype 3 infection and the autoimmune background.

The derivation of human brain capillary endothelial cells is of utmost importance for drug discovery programs focusing on diseases of the central nervous system. Here, we describe a two-step differentiation protocol to derive brain capillary-like endothelial cells from human pluripotent stem cells. The cells were initially differentiated into endothelial progenitor cells followed by specification into a brain capillary-like endothelial cell phenotype using a protocol that combined the induction, in a time-dependent manner, of VEGF, Wnt3a, and retinoic acid signaling pathways and the use of fibronectin as the extracellular matrix. The brain capillary-like endothelial cells displayed a permeability to lucifer yellow of 1 × 10 -3 cm/min, a transendothelial electrical resistance value of 60 Ω cm 2 and were able to generate a continuous monolayer of cells expressing ZO-1 and CLAUDIN-5 but moderate expression of P-glycoprotein. Further maturation of these cells required coculture with pericytes. The study presented here opens a new approach for the study of soluble and non-soluble factors in the specification of endothelial progenitor cells into brain capillary-like endothelial cells., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Here we report a two-step surface modification methodology to radiolabel small extracellular vesicles (SEVs) with 64CuCl2 for PET/MRI imaging. The modification did not change or damage the morphology, surface receptor proteins and internal RNA content. Radiolabeled SEVs could be detected in organs with low accumulation such as the brain (0.4-0.5% ID g-1) and their brain location determined by MRI.

Sacubitril/valsartan has been shown to improve clinical outcomes in patients with heart failure and reduced ejection fraction (HFrEF), but its effects on left ventricular (LV) systolic function and reverse remodeling parameters remain to be established. We hereby describe the case of a 41 year old man with HFrEF and severe reduction of left ventricular ejection fraction (LVEF). The patient was first treated with triple HF therapy (beta-blocker, angiotensin converting enzyme inhibitor and mineralocorticoid antagonist), but after three months he was still symptomatic and with an LVEF firmly low. In consideration of poor response to therapy, we switched angiotensin converting enzyme inhibitor to sacubitril/valsartan to the maximum tolerated dose (49/51 mg bid) with a marked improvement in LV systolic function and reduction in LV volumes at follow-up. In light of the almost normalized LVEF the patient was also removed from the list for the implantation of a cardiac resynchronization therapy defibrillator. In conclusion, our case showed a strong beneficial effect of sacubitril/valsartan on reverse remodeling and LVEF beyond the benefits of concomitant optimal medical therapy. This result is particular noteworthy because it was obtained although the patient wasn't able to reach the full dose of the drug. Physicians should always consider this drug effect when more demanding therapeutic strategies are needed for their HF patients.

Objective: To characterize the profile of non-AIDS-related comorbidities (NARC) in the older HIV-1-infected population and to explore the factors associated with multiple NARC., Methods: This was a multicentre, cross-sectional study including HIV-1-infected patients aged ≥50 years, who were virologically suppressed and had been on a stable antiretroviral therapy (ART) regimen for at least 6 months. A multiple regression model explored the association between demographic and clinical variables and the number of NARC., Results: Overall, 401 patients were enrolled. The mean age of the patients was 59.3 years and 72.6% were male. The mean duration of HIV-1 infection was 12.0 years and the median exposure to ART was 10.0 years. The mean number of NARC was 2.1, and 34.7% of patients had three or more NARC. Hypercholesterolemia was the most frequent NARC (60.8%), followed by arterial hypertension (39.7%) and chronic depression/anxiety (23.9%). Arterial hypertension and diabetes mellitus were the most frequently treated NARC (95.6% and 92.6% of cases, respectively). The linear regression analysis showed a positive relationship between age and NARC (B=0.032, 95% confidence interval 0.015-0.049; p=0.0003) and between the duration of HIV-1 infection and NARC (B=0.039, 95% confidence interval 0.017-0.059; p=0.0005)., Conclusions: A high prevalence of NARC was found, the most common being metabolic, cardiovascular, and psychological conditions. NARC rates were similar to those reported for the general population, suggesting a larger societal problem beyond HIV infection. A multidisciplinary approach is essential to reduce the burden of complex multi-morbid conditions in the HIV-1-infected population., (Copyright © 2018 Merck Sharp & Dohme Corp and The Authors. Published by Elsevier Ltd.. All rights reserved.)

Stimulation of adult neurogenesis by targeting the endogenous neural stem cells (NSCs), located in hippocampus and subventricular zone (SVZ), with nanoformulations has been proposed for brain repair in cases of neurodegenerative diseases. Unfortunately, it is relatively unknown the nanoformulation properties to facilitate their accumulation in the neurogenic niches after intravenous injection. Here, we have screened different gold-based formulations having variable morphology, surface chemistry and responsiveness to light for their capacity to cross the blood brain barrier (BBB) and accumulate preferentially in the neurogenic niches. Results obtained in a human in vitro BBB model showed that gold nanoparticles (Au NPs) and gold nanorods (Au NRs) conjugated with medium density of transferrin (Tf) peptides (i.e. between 169 and 230 peptides per NP) crossed more efficiently the BBB than the remaining formulations. This is due to a relatively lower avidity of these formulations to Tf receptor (TfR) and lower accumulation in the lysosomes, as compared to the other formulations. We further show that the near infrared light (NIR) irradiation of Au NRs, under a certain concentration and at specific cell culture time, lead to the opening of the BBB. Finally, we demonstrate that Au NRs conjugated with Tf administered intravenously in mice and activated by NIR had the highest accumulation in the neurogenic niches. Our results open the possibility of targeting more effectively the neurogenic niches by controlling the properties of the nanoformulations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepatitis E in industrialized countries is mainly associated with genotype 3 hepatitis E virus (HEV) and normally causes a sporadic self-limiting disease in immunocompetent individuals. Unlike genotype 3, genotypes 1 and 2 circulate in developing countries, produce severe disease and occur in the epidemic form. Hepatitis E occurring in travellers returning from endemic areas in developing countries is not a novel epidemiological occurrence, however the vast majority of cases remain to be genetically studied. The present study describes two cases of severe acute hepatitis E that required hospitalization for 6 and 9 days in two individuals of Indian nationality that had recently migrated to Portugal to work. The retrieved HEV sequences both belonged to genotype 1 and had a high degree of nucleotide sequence identity, clustering with strains isolated in India and Nepal, in 2013 and 2014. Confirmed HEV genotypes of increased pathogenicity like genotype 1 are being introduced into otherwise naïve populations of industrialized countries such as European countries with consequences difficult to predict. As far as we know the present study is the first in Portugal to describe and genetically characterize imported cases of hepatitis E infection caused by HEV genotype 1.

Background: Case report, in a patient with a history of diabetes and hypertension, treated with metformin, gliclazide, enalapril + hydrochlorothiazide, amlodipine, aspirin and diazepam, recently medicated for a gouty crisis with colchicine and clonixin without improvement. Believing it could help in the treatment of gouty crisis symptoms he took about 1.5 L of artichoke infusion (Cynara cardunculus). He felt better and did agriculture work but developed a distal muscle pain, severe anemia, standard biochemical liver cholestasis, increase of alkaline phosphatase and marked increase of inflammatory parameters (hyperleucocytosis) and enters in the emergency department at the hospital., Objective: Evaluation of the cause of complaints and laboratory abnormalities and the involvement of artichoke infusion., Results: The prominence of the inflammatory parameters was ruled out because of exhaustive autoimmune, infectious or para-neoplastic syndrome (blood cultures, serology, diagnostic imaging, bone marrow and bone biopsy, muscle biopsy and nerve, abdominal angiography) were carried out showing normal results. The evaluation pointed out that the concomitant intake of artichoke infusion may have been involved in the framework developed, since the drugs which were being administered to/by the patient have a metabolism mainly mediated by CYP450 3A4 and 2C9 that could be compromised when these isoenzymes are inhibited by phenolic and flavonoid compounds from plants. Colchicine was one of the last drugs took that have as side effects most of the symptoms felt by patient including diarrhea and anemia., Conclusion: The spontaneous and complete recovery of the patient and the negativity of research looking for other causes, conduce to a strong possibility of the interaction between artichoke and the drugs in the clinical presentation of this case., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Wound treatment remains one of the most prevalent and economically burdensome healthcare issues in the world. Poly (lactic-co-glycolic acid) (PLGA) supplies lactate that accelerates neovascularization and promotes wound healing. LL37 is an endogenous human host defense peptide that modulates wound healing and angiogenesis and fights infection. Hence, we hypothesized that the administration of LL37 encapsulated in PLGA nanoparticles (PLGA-LL37 NP) promotes wound closure due to the sustained release of both LL37 and lactate. In full thickness excisional wounds, the treatment with PLGA-LL37 NP significantly accelerated wound healing compared to PLGA or LL37 administration alone. PLGA-LL37 NP-treated wounds displayed advanced granulation tissue formation by significant higher collagen deposition, re-epithelialized and neovascularized composition. PLGA-LL37 NP improved angiogenesis, significantly up-regulated IL-6 and VEGFa expression, and modulated the inflammatory wound response. In vitro, PLGA-LL37 NP induced enhanced cell migration but had no effect on the metabolism and proliferation of keratinocytes. It displayed antimicrobial activity on Escherichia coli. In conclusion, we developed a biodegradable drug delivery system that accelerated healing processes due to the combined effects of lactate and LL37 released from the nanoparticles., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: This cross-sectional study investigated whether the theory of planned behavior (TPB) constructs: attitudes, subjective norms, and perceived behavioral control were related to intention of dietary supplements use among African-American women living with Human Immunodeficiency Virus and/or Acquired Immune Deficiency Syndrome (HIV/AIDS)., Methods: A closed-ended questionnaire based on the TPB was utilized to explore the use of dietary supplements among a cohort of 153 HIV-positive African-American women., Results: Overall, 45% of the respondents used dietary supplements to manage/control their HIV. Combined, attitudes, subjective norms and perceived behavioral control were significant predictors of intention toward dietary supplement use (69% of the variance explained, p<0.0001). Attitudes (β=0.23, p<0.001) and perceived behavioral control (β=0.45, p<0.0001) were found to be significant independent predictors of intention. Behavioral intention and proximal TPB constructs (attitudes, subjective norms, and perceived behavioral control), as well as their underlying beliefs about dietary supplements use, were all found to be significantly more positive in users of dietary supplements compared to non-users (p<0.001)., Conclusions: Results showed that attitudes, subjective norms and perceived behavioral control are important predictors in the intention to use dietary supplements for control of HIV among African-American women. Implications from this study suggest that the TPB can be used to better identify and understand salient beliefs that surround intentions to use alternative therapies for management of disease. These beliefs can be used to develop interventions surrounding HIV treatment and care., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The use of tissue adhesives for internal clinical applications is limited due to a lack of materials that balance strong adhesion with biocompatibility. The use of substrate topography is explored to reduce the volume of a highly reactive and toxic glue without compromising adhesive strength. Micro-textured patches coated with a thin layer of cyanoacrylate glue achieve similar adhesion levels to patches employing large amounts of adhesive, and is superior to the level of adhesion achieved when a thin coating is applied to a non-textured patch. In vivo studies demonstrate reduced tissue inflammation and necrosis for patterned patches with a thinly coated layer of reactive glue, thus overcoming a significant challenge with existing tissue adhesives such as cyanoacrylate. Closure of surgical stomach and colon defects in a rat model is achieved without abdominal adhesions. Harnessing the synergy between surface topography and reactive chemistry enables controlled tissue adhesion with an improved biocompatibility profile without requiring changes in the chemical composition of reactive tissue glues., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Currently, there are no clinically approved surgical glues that are nontoxic, bind strongly to tissue, and work well within wet and highly dynamic environments within the body. This is especially relevant to minimally invasive surgery that is increasingly performed to reduce postoperative complications, recovery times, and patient discomfort. We describe the engineering of a bioinspired elastic and biocompatible hydrophobic light-activated adhesive (HLAA) that achieves a strong level of adhesion to wet tissue and is not compromised by preexposure to blood. The HLAA provided an on-demand hemostatic seal, within seconds of light application, when applied to high-pressure large blood vessels and cardiac wall defects in pigs. HLAA-coated patches attached to the interventricular septum in a beating porcine heart and resisted supraphysiologic pressures by remaining attached for 24 hours, which is relevant to intracardiac interventions in humans. The HLAA could be used for many cardiovascular and surgical applications, with immediate application in repair of vascular defects and surgical hemostasis.

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., (Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

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-1β 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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

Recent pre-clinical and clinical studies indicate that certain exogenous stem cells and biomaterials can preserve cardiac tissue after myocardial infarction. Regarding stem cells, a growing body of data suggests that the short-term positive outcomes are mainly attributed to paracrine signaling mechanisms. The release of such factors is due to the cell's ability to sense cardiac environmentally derived cues, though the exact feedback loops are still poorly understood. However, given the limited engraftment and survival of transplanted cells in the ischemic environment, the long-term clinical benefits of these therapies have not yet been realized. To overcome this, the long-term controlled delivery of bioactive factors using biomaterials is a promising approach. A major challenge has been the ability to develop timely and spatially controlled gradients of different cues, pivotal for the development and regeneration of tissues. In addition, given the complexity of the remodeling process after myocardial infarction, multiple factors may be required at distinct disease stages to maximize therapeutic outcomes. Therefore, novel smart materials that can sense the surrounding environment and generate cues through on demand mechanisms will be of major importance in the translation of these promising advanced therapies. This article reviews how the cardiac environment can mediate the release profiles of bioactive cues from cells and biomaterials and how the controlled delivery impacts heart regeneration.

The self-renewal and differentiation of human pluripotent stem cells (hPSCs) have typically been studied in flat, two-dimensional (2D) environments. In this Perspective, we argue that 3D model systems may be needed in addition, as they mimic the natural 3D tissue organization more closely. We survey methods that have used 3D biomaterials for expansion of undifferentiated hPSCs, directed differentiation of hPSCs and transplantation of differentiated hPSCs in vivo.

We present use of a synthetic, injectable matrix metalloproteinase (MMP)-responsive, bioactive hydrogel as an in situ forming scaffold to deliver thymosin β4 (Tβ4), a pro-angiogenic and pro-survival factor, along with vascular cells derived from human embryonic stem cells (hESC) in ischemic injuries to the heart in a rat model. The gel was found to substitute the degrading extracellular matrix in the infarcted myocardium of rats and to promote structural organization of native endothelial cells, while some of the delivered hESC-derived vascular cells formed de novo capillaries in the infarct zone. Magnetic resonance imaging (MRI) revealed that the microvascular grafts effectively preserved contractile performance 3 d and 6 wk after myocardial infarction, attenuated left ventricular dilation, and decreased infarct size as compared to infarcted rats treated with PBS injection as a control (3 d ejection fraction, + ∼7%, P < 0.001; 6 wk ejection faction, + ∼12%, P < 0.001). Elevation in vessel density was observed in response to treatment, which may be due in part to elevations in human (donor)-derived cytokines EGF, VEGF and HGF (1 d). Thus, a clinically relevant matrix for dual delivery of vascular cells and drugs may be useful in engineering sustained tissue preservation and potentially regenerating ischemic cardiac tissue., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

Herein, we describe a protocol for the isolation of human embryonic stem cells (hESCs)-derived vascular cells at various stages of development. The cells are isolated from 10 to 15-d-old human embryoid bodies (EBs) cultured in suspension. After dissociation, cells are labeled with anti-CD34 or anti-CD31 (PECAM1) antibody and separated from the cell mixture by magnetic-activated cell separation (MACS) or fluorescent-activated cell sorting (FACS). Isolated vascular cells are then cultured in media conditions that support specific differentiation and expansion pathways. The resulting vascular cell populations contain >80% endothelial-like or smooth muscle-like cells. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 1.5 months. Vascular cells isolated and differentiated under the described conditions may constitute a potential cell source for therapeutic application toward repair of ischemic tissues, preparation of tissue-engineered vascular grafts and design of cellular kits for drug screening applications.

The vascularization of tissue constructs remains a major challenge in regenerative medicine, as the diffusional supply of oxygen can support only 100-200 mum thick layers of viable tissue. The formation of a mature and functional vascular network requires communication between endothelial cells (ECs) and smooth muscle cells (SMCs). Potential sources of these cells that involve noninvasive methodologies are required for numerous applications including tissue-engineered vascular grafts, myocardial ischemia, wound healing, plastic surgery, and general tissue-engineering applications. Human embryonic stem cells (hESCs) can be an unlimited source of these cells. They can be expanded in vitro in an undifferentiated state without apparent limit, and hES-derived cells can be created in virtually unlimited amounts for potential clinical uses. Recently, vascular progenitor cells as well as endothelial and smooth muscle cells have been isolated from hESCs.

The in vitro potential of a synthetic matrix metalloproteinase (MMP)-responsive poly(ethylene glycol) (PEG)-based hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, thymosin beta4 (Tbeta4), was examined. We show that the physical incorporation of Tbeta4 in this bioactive matrix creates a three-dimensional (3D) environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped Tbeta4 increased the survival of HUVEC compared to gels without Tbeta4, and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas von Willebrand factor was significantly down-regulated. Incorporation of Tbeta4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC. The gel acts as a controlled Tbeta4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, Tbeta4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues.

Highly fluorescent core-shell silica nanoparticles made by the modified Stöber process (C dots) are promising as tools for sensing and imaging subcellular agents and structures but will only be useful if they can be easily delivered to the cytoplasm of the subject cells. This work shows that C dots can be electrostatically coated with cationic polymers, changing their surface charge and enabling them to escape from endosomes and enter the cytoplasm and nucleus. As an example of cellular delivery, we demonstrate that these particles can also be complexed with DNA and mediate and trace DNA delivery and gene expression.

We report that human embryonic stem cells contain a population of vascular progenitor cells that have the ability to differentiate into endothelial-like and smooth muscle (SM)-like cells. Vascular progenitor cells were isolated from EBs grown in suspension for 10 days and were characterized by expression of the endothelial/hematopoietic marker CD34 (CD34+ cells). When these cells are subsequently cultured in EGM-2 (endothelial growth medium) supplemented with vascular endothelial growth factor-165 (50 ng/mL), they give rise to endothelial-like cells characterized by a cobblestone cell morphology, expression of endothelial markers (platelet endothelial cell-adhesion molecule-1, CD34, KDR/Flk-1, vascular endothelial cadherin, von Willebrand factor), incorporation of acetylated low-density lipoprotein, and formation of capillary-like structures when placed in Matrigel. In contrast, when CD34+ cells are cultured in EGM-2 supplemented with platelet-derived growth factor-BB (50 ng/mL), they give rise to SM-like cells characterized by spindle-shape morphology, expression of SM cell markers (alpha-SM actin, SM myosin heavy chain, calponin, caldesmon, SM alpha-22), and the ability to contract and relax in response to common pharmacological agents such as carbachol and atropine but rarely form capillary-like structures when placed in Matrigel. Implantation studies in nude mice show that both cell types contribute to the formation of human microvasculature. Some microvessels contained mouse blood cells, which indicates functional integration with host vasculature. Therefore, the vascular progenitors isolated from human embryonic stem cells using methods established in the present study could provide a means to examine the mechanisms of endothelial and SM cell development, and they could also provide a potential source of cells for vascular tissue engineering.

Control of self-renewal and differentiation of human ES cells (hESCs) remains a challenge. This is largely due to the use of culture systems that involve poorly defined animal products and do not mimic the normal developmental milieu. Routine protocols involve the propagation of hESCs on mouse fibroblast or human feeder layers, enzymatic cell removal, and spontaneous differentiation in cultures of embryoid bodies, and each of these steps involves significant variability of culture conditions. We report that a completely synthetic hydrogel matrix can support (i) long-term self-renewal of hESCs in the presence of conditioned medium from mouse embryonic fibroblast feeder layers, and (ii) direct cell differentiation. Hyaluronic acid (HA) hydrogels were selected because of the role of HA in early development and feeder layer cultures of hESCs and the controllability of hydrogel architecture, mechanics, and degradation. When encapsulated in 3D HA hydrogels (but not within other hydrogels or in monolayer cultures on HA), hESCs maintained their undifferentiated state, preserved their normal karyotype, and maintained their full differentiation capacity as indicated by embryoid body formation. Differentiation could be induced within the same hydrogel by simply altering soluble factors. We therefore propose that HA hydrogels, with their developmentally relevant composition and tunable physical properties, provide a unique microenvironment for the self-renewal and differentiation of hESCs.

We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF(165). The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including alpha-fetoprotein decreased 2- or 3-fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine.

Osteogenic cultures of embryonic stem cells (ESCs) are predominately derived from three-dimensional cell spheroids called embryoid bodies (EBs). An alternative method that has been attempted and merits further attention avoids EBs through the immediate separation of ESC colonies into single cells. However, this method has not been well characterized and the effect of omitting the EB step is unknown. Herein, we report that culturing human embryonic stem cells (hESCs) without the EB stage leads to a sevenfold greater number of osteogenic cells and to spontaneous bone nodule formation after 10-12 days. In contrast, when hESCs were differentiated as EBs for 5 days followed by plating of single cells, bone nodules formed after 4 weeks only in the presence of dexamethasone. Furthermore, regardless of the inclusion of EBs, bone matrix formed, including cement line matrix and mineralized collagen, which displayed apatitic mineral (PO4) with calcium-to-phosphorous ratios similar to those of hydroxyapatite and human bone. Together these results demonstrate that culturing hESCs without an EB step can be used to derive large quantities of functional osteogenic cells for bone tissue engineering.

In order to test the hypothesis that transforming growth factor beta (TGF-beta) acts by FS regulation on bovine granulosa cells in in vitro differentiation, we analyzed the effect of TGF-beta1 on follistatin mRNA expression in three differentiation states of bovine granulosa cells. We showed a positive regulation of FS mRNA after TGF-beta1 (1 ng/ml) treatment of freshly isolated granulosa cells from small-medium antral follicles (2-8 mm). This effect was abolished by the addition of exogenous follistatin (100 ng/ml), suggesting that this effect could be mediated by activin. Although these cells showed a similar effect on FS mRNA expression after treatment with activin-A, a soluble form of activin receptor type IIA was unable to inactivate the TGF-beta effect. When we tested the TGF-beta effect on FS mRNA in different granulosa cell states, TGF-beta1 regulation was associated with progesterone production only in freshly isolated cells. The amount of total activin-A produced by first passage cells (dedifferentiated cells), was ten times smaller than the one measured in a conditioned medium from freshly isolated cells (mature cells). The TGF-beta1-dependent FS mRNA expression persisted in first passage cells without changes with FS addition. On the other hand, the BGC-1 granulosa cell line (immature cells) produced large amounts of activin-A regulated by TGF-beta1 and an invariable steady state of FS mRNAs. In summary, our results showed that FS mRNA expression is regulated by TGF-beta1 independently of activin effects in differentiated granulosa cells.

Knowledge of population ancestry from genetic markers is essential, for example, to understand the history of human migration and to carry out admixture and association studies. Here we assess the genome ancestry of the Azorean population through analysis of six Alu polymorphic sites (TPA-25, ACE, APO, B65, PV92, and D1) in 65 Azoreans and 30 Portuguese unrelated blood donors and compare data for the Y-chromosome and mtDNA. Allele frequencies were calculated by direct counting. Statistical analysis was performed using Arlequin 2.0. Nei's genetic distance was calculated with DISPAN software, and trees were constructed by neighbor joining (NJ) using PHYLIP 3.63. The results show that all Alu insertions were polymorphic. APO is the closest to fixation. The less frequent insertions are PV92 and D1 in the Azores and Portugal, respectively. ACE and TPA-25 show the highest values of heterozygosity in both populations. Allele frequencies are very similar to those obtained in European populations. These results are validated by the Y-chromosome and mtDNA data, where the majority of the maternal and paternal lineages are European. Overall, these data are reflected in the phylogenetic tree, in which the Azoreans and the Portuguese branch with Catalans, Andalusians, Moroccans, and Algerians. We conclude that the population of the Azores shows no significant genetic differences from that of mainland Portugal and that it is an outbred population. Moreover, the data validate the use of Alu insertion polymorphisms to assess the origin and history of human populations., (Am. J. Hum. Biol. 18:223-226, 2006. (c) 2006 Wiley-Liss, Inc.)

The first clinical case described in Portugal of a human infection by Rhodococcus equi in a 35 year-old HIV1 seropositive i.v. drug user is presented. The patient was admitted to hospital due to clinical and radiological suspicion of tuberculosis. While in hospital, a Rhodococcus equi cavitated lung infection was diagnosed. By applying a triple antibiotic therapy with erythromycin, rifampin and teicoplanin along with surgical excision of the lower lobe of the right lung, we were able to clinically resolve the pulmonary infection. A brief review of Rhodococcus equi infections, especially those associated to HIV1 infected individuals is also presented with particular emphasis on the therapeutic approach.

Fewer than one third of patients presenting to the emergency department with complaints of chest pain have an acute coronary syndrome. The electrocardiogram provides a specific diagnosis only in 40% of patients with acute myocardial infarction. The presence of regional wall-motion abnormalities at echocardiography in patients without known coronary artery disease is a moderate indicator of an increased likelihood of acute myocardial ischemia or myocardial infarction with a positive predictive accuracy of about 50%. More important, the absence of regional wall-motion abnormalities identifies a subset of patients unlikely to have a myocardial infarction with a negative predictive accuracy of about 95%. Echocardiography can provide incremental prognostic information to identify patients at risk of early or late cardiac events, even after consideration of clinical, historical, and electrocardiographic variables. The application of new contrast agents to echocardiography will probably allow an early and more accurate evaluation of patients with chest pain of uncertain significance.

The interactions between respiration, heart rate and blood pressure variability (HRV, BPV), are considered to be of paramount importance for the study of the functional organisation of the autonomic nervous system (ANS). The aim of the reported study is to detect and classify the intermittent phase locking (PL) phenomena between respiration, HRV and BPV during cardiorespiratory synchronisation experiments, by using the following time-domain techniques: Poincaré maps, recurrence plots, time-space separation plots and frequency tracking locus. The experimental protocol consists of three stages, with normal subjects in paced breathing at 15, 12 and 8 breaths min-1. Transient phenomena of coordination between respiration and the major rhythms of HRV and BPV (low and high frequency, LF and HF) have been detected and classified: no interaction between LF and HF rhythms at 15 breaths min-1; short time intervals of stable 1:2 frequency and phase synchronisation during the 12 breaths min-1 stage; 1:1 PL during the 8 breaths min-1 stage. 1:1 and 1:2 PL phenomena occurred when the respiration frequency was quite close to the LF frequency or when it was about twice the LF frequency, respectively. The complex organisation of the ANS seems to provoke transient rather than permanent PL phenomena between the co-ordinating components of respiration and cardiovascular variability series.

Aim of this study is to assess whether and to what extent LF and HF rhythms contribute to spontaneous pupil diameter fluctuations at rest and during sympathetic activation by Head-Up Tilt Test 70 degrees. ECG, Finapres and Respiration data from 10 normal subjects were recorded simultaneously to spontaneous pupil diameter fluctuations (SPDF). The respiratory rhythm and the LF component were clearly detected in all the SPDF series. The cross-spectrum analysis gave significant results (> 0.5) in HF band. During tilt, a reduction in the total variability with a relative increase of the LF component was observed in HR, BP and SPDF signals. The multivariate analysis of the SPDF and of the cardiovascular variability series allows new insights into the physiology of pupil neural control. The effect of tilting on SPDF spectra and the physiological background of the pupil control are consistent with the hypothesis that pupil fluctuations reflect a generalized increase of the sympathetic tone.

Studies using spectral analysis of cardiovascular variability series, as a non invasive tool for assessing the autonomic nervous system activity, have attracted growing interest in the last 20 years. Short-term recordings of heart rate variability distinguish two main spectral components: a high-frequency (HF) component (ranging between 0.15-0.40 Hz), and a low-frequency (LF) component (ranging between 0.04-0.15 Hz), respectively considered markers of parasympathetic and sympathetic control. Spectral analysis of microcirculatory blood flow, by using laser Doppler flowmetry, recently disclosed the presence of similar rhythms. Although a general agreement about the amplitude and frequency of these spectral components has not been gained yet, evidences of an autonomic nervous system role have been collected. Also the pupil diameter spontaneously fluctuates in the HF range. The aim of this study was to design an experimental set up for the simultaneous and continuous recording of heart rate, blood pressure, respiratory activity, peripheral blood flow, and pupil diameter fluctuations in order to investigate whether common spontaneous rhythms could be detected into the variability series. We enrolled 10 normal volunteers (range 24-28 years). The variability series of heart rate, blood pressure, respiratory activity, peripheral blood flow and pupil diameter were obtained at rest and during sympathetic activation by head-up tilt test 70 degrees. Subjects were instructed to breath at 15 breath/min following an audio signal and adjusting their tidal volume to a comfortable level. Peripheral blood flow was non invasively monitored with laser Doppler technique, with the two probes located in the palmar site of the hand and in the sternum. We continuously assess the pupil diameter fluctuations by a custom, portable, infrared TV pupillometer. Spectral density was estimated by autoregressive modeling on 250-point segments. The respiratory rhythm was clearly detected in all the variability series. Oscillations in the LF band were detected in heart rate, blood pressure and in the laser Doppler signals of the hand, as well as in the pupil diameter, but not always in the laser Doppler flow of the sternum. In this last signal an harmonic component at 0.16-0.17 Hz was detected. A reduction in the total variability, with a relative increase in the LF component was observed during head-up tilt test in the heart rate, blood pressure, peripheral blood flow and pupil diameter signals. These findings are consistent with the increased sympathetic modulation induced by the head-up tilt test. These data confirm previous findings and observations, collected with different techniques and for different purposes. Whether these rhythms can be expression of central common oscillators or reflex mechanical factors is of primary importance for possible clinical applications of this approach.

We studied a possible correlation between autonomic cardiac activity and the level of the red blood cell acetylcholinesterase (AChE) in patients with probable Alzheimer disease (AD). The influence of cholinesterase inhibitor treatment on this autonomic activity was evaluated. Twelve patients satisfying the NINCDS-ADRDA criteria of probable AD and 10 healthy controls were studied. Autonomic cardiac activity was evaluated by means of power spectral analysis (PSA) of heart rate variability (HRV) using an autoregressive algorithm on 250 consecutive electrocardiographic R-R intervals. All patients received oral eptastigmine, a new cholinesterase inhibitor, for 1 month. Before treatment, a simultaneous recording of the electrocardiographic and respiratory activities was performed at rest and subsequently during head-up tilt test at 700. Recording was repeated on the last day of treatment. The level of AChE activity during each recording was also evaluated. Spectrum power was calculated in three main frequency bands: high frequency (HF), 0.15-0.4 Hz; low frequency (LF), 0.04-0.15 Hz; very low frequency (VLF), <0.04 Hz. In addition, we calculated the total spectrum power (TSP) and the LF/HF ratio. The TSP and the absolute value of each spectral component were significantly lower in AD patients than in controls. In contrast with controls, AD patients did not show any significant change before treatment in either the LF and HF components or in the LF/HF ratio during the tilt test. However, the modification in the LF component, induced by tilting, showed a significant correlation with the level of AChE activity (p < 0.03). During the tilt test, the treatment caused changes in LF and HF components and in the LF/HF ratio similar to those observed in controls. These results suggest that the presence of autonomic cardiac dysfunction in AD patients might be due to a cholinergic deficit in the peripheral autonomic nervous system. Some aspects of this autonomic dysfunction can be normalized by cholinesterase inhibitor treatment.

No data are available on the autonomic system during sleep in patients with stroke. The purpose of this study was to determine the influence of acute ischemic stroke on the autonomic cardiovascular system during sleep, and to correlate autonomic activity with the clinical status of patients. Ten patients with ischemic stroke in the middle cerebral artery were studied by means of an all-night polysomnographic recording within the 1st week of the onset of symptoms and at the 3-week follow-up examination. Power spectrum analysis of the heart rate variability was performed using an autoregressive algorithm in 180 consecutive electrocardiographic RR intervals. Spectral power was calculated in 3 main frequency bands: high frequency (HF), 0.15-0.4 Hz; low frequency (LF), 0.04-0.15 Hz; very low frequency (VLF), < 0.04 Hz. The data were compared with those of 10 age-matched controls. A significant increase in VLF (p < 0.0005) and a decrease in HF (p < 0.0002) components were found in ischemic stroke patients. The sympathetic-parasympathetic balance (VLF + LF/HF) was higher in patients than controls (p < 0.005). However, these components changed significantly during sleep, revealing a physiological pattern. These power spectral data were still present at the 3-week follow-up. The 4 patients who developed cardiac arrhythmias showed higher sympathetic-parasympathetic balance than patients without arrhythmias (p < 0.05). These data suggest a sympathetic predominance in patients with acute ischemic stroke during sleep. However, the flexible and dynamic properties of the autonomic nervous system are preserved. Cardiac arrhythmias following stroke may be related to the degree of sympathetic predominance.