Development And Characterization Of A Cationic Lipid Nanocarrier As Non-viral Vector For Gene Therapy.

The aim of the present work was to produce a cationic solid lipid nanoparticle (SLN) as non-viral vector for protein delivery. Cationic SLN were produced by double emulsion method, composed of softisan(®) 100, cetyltrimethylammonium bromide (CTAB), Tween(®) 80, Span(®) 80, glycerol and lipoid(®) S75 loading insulin as model protein. The formulation was characterized in terms of mean hydrodynamic diameter (z-ave), polydispersity index (PI), zeta potential (ZP), stability during storage time, stability after lyophilization, effect of toxicity and transfection ability in HeLa cells, in vitro release profile and morphology. SLN were stable for 30days and showed minimal changes in their physicochemical properties after lyophilization. The particles exhibited a relatively slow release, spherical morphology and were able to transfect HeLa cells, but toxicity remained an obstacle. Results suggest that SLN are nevertheless promising for delivery of proteins or nucleic acids for gene therapy.

Evaluation Of Red Cell And Reticulocyte Parameters As Indicative Of Iron Deficiency In Patients With Anemia Of Chronic Disease.

The purpose of this study was to evaluate the effectiveness of mature red cell and reticulocyte parameters under three conditions: iron deficiency anemia, anemia of chronic disease, and anemia of chronic disease associated with absolute iron deficiency. Peripheral blood cells from 117 adult patients with anemia were classified according to iron status, and inflammatory activity, and the results of a hemoglobinopathy investigation as: iron deficiency anemia (n=42), anemia of chronic disease (n=28), anemia of chronic disease associated with iron deficiency anemia (n=22), and heterozygous β thalassemia (n=25). The percentage of microcytic red cells, hypochromic red cells, and levels of hemoglobin content in both reticulocytes and mature red cells were determined. Receiver operating characteristic analysis was used to evaluate the accuracy of the parameters in differentiating between the different types of anemia. There was no significant difference between the iron deficient group and anemia of chronic disease associated with absolute iron deficiency in respect to any parameter. The percentage of hypochromic red cells was the best parameter to discriminate anemia of chronic disease with and without absolute iron deficiency (area under curve=0.785; 95% confidence interval: 0.661-0.909, with sensitivity of 72.7%, and specificity of 70.4%; cut-off value 1.8%). The formula microcytic red cells minus hypochromic red cells was very accurate in differentiating iron deficiency anemia and heterozygous β thalassemia (area under curve=0.977; 95% confidence interval: 0.950-1.005; with sensitivity of 96.2%, and specificity of 92.7%; cut-off value 13.8). The indices related to red cells and reticulocytes have a moderate performance in identifying absolute iron deficiency in patients with anemia of chronic disease.

Apoplastic And Intracellular Plant Sugars Regulate Developmental Transitions In Witches' Broom Disease Of Cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

Tshr Intronic Polymorphisms (rs179247 And Rs12885526) And Their Role In The Susceptibility Of The Brazilian Population To Graves' Disease And Graves' Ophthalmopathy.

Intronic thyroid-stimulating hormone receptor polymorphisms have been associated with the risk for both Graves' disease and Graves' ophthalmopathy, but results have been inconsistent among different populations. We aimed to investigate the influence of thyroid-stimulating hormone receptor intronic polymorphisms in a large well-characterized population of GD patients. We studied 279 Graves' disease patients (231 females and 48 males, 39.80 ± 11.69 years old), including 144 with Graves' ophthalmopathy, matched to 296 healthy control individuals. Thyroid-stimulating hormone receptor genotypes of rs179247 and rs12885526 were determined by Real Time PCR TaqMan(®) SNP Genotyping. A multivariate analysis showed that the inheritance of the thyroid-stimulating hormone receptor AA genotype for rs179247 increased the risk for Graves' disease (OR = 2.821; 95 % CI 1.595-4.990; p = 0.0004), whereas the thyroid-stimulating hormone receptor GG genotype for rs12885526 increased the risk for Graves' ophthalmopathy (OR = 2.940; 95 % CI 1.320-6.548; p = 0.0083). Individuals with Graves' ophthalmopathy also presented lower mean thyrotropin receptor antibodies levels (96.3 ± 143.9 U/L) than individuals without Graves' ophthalmopathy (98.3 ± 201.9 U/L). We did not find any association between the investigated polymorphisms and patients clinical features or outcome. We demonstrate that thyroid-stimulating hormone receptor intronic polymorphisms are associated with the susceptibility to Graves' disease and Graves' ophthalmopathy in the Brazilian population, but do not appear to influence the disease course.

The Ccr5Δ32 Polymorphism In Brazilian Patients With Sickle Cell Disease.

Previous studies on the role of inflammation in the pathophysiology of sickle cell disease (SCD) suggested that the CCR5Δ32 allele, which is responsible for the production of truncated C-C chemokine receptor type 5 (CCR5), could confer a selective advantage on patients with SCD because it leads to a less efficient Th1 response. We determined the frequency of the CCR5Δ32 polymorphism in 795 Afro-Brazilian SCD patients followed up at the Pernambuco Hematology and Hemotherapy Center, in Northeastern Brazil, divided into a pediatric group (3 months-17 years, n = 483) and an adult group (18-70 years, n = 312). The adult patients were also compared to a healthy control group (blood donors, 18-61 years, n = 247). The CCR5/CCR5Δ32 polymorphism was determined by allele-specific PCR. No homozygous patient for the CCR5Δ32 allele was detected. The frequency of heterozygotes in the study population (patients and controls) was 5.8%, in the total SCD patients 5.1%, in the children 5.4%, in the adults with SCD 4.8%, and in the adult controls 8.1%. These differences did not reach statistical significance. Our findings failed to demonstrate an important role of the CCR5Δ32 allele in the population sample studied here.

Perianal Complete Remission With Combined Therapy (seton Placement And Anti-tnf Agents) In Crohn's Disease: A Brazilian Multicenter Observational Study.

Perianal fistulizing Crohn's disease is one of the most severe phenotypes of inflammatory bowel diseases. Combined therapy with seton placement and anti-TNF therapy is the most common strategy for this condition. The aim of this study was to analyze the rates of complete perianal remission after combined therapy for perianal fistulizing Crohn's disease. This was a retrospective observational study with perianal fistulizing Crohn's disease patients submitted to combined therapy from four inflammatory bowel diseases referral centers. We analyzed patients' demographic characteristics, Montreal classification, concomitant medication, classification of the fistulae, occurrence of perianal complete remission and recurrence after remission. Complete perianal remission was defined as absence of drainage from the fistulae associated with seton removal. A total of 78 patients were included, 44 (55.8%) females with a mean age of 33.8 (±15) years. Most patients were treated with Infliximab, 66.2%, than with Adalimumab, 33.8%. Complex fistulae were found in 52/78 patients (66.7%). After a medium follow-up of 48.2 months, 41/78 patients (52.6%) had complete perianal remission (95% CI: 43.5%-63.6%). Recurrence occurred in four (9.8%) patients (95% CI: 0.7%-18.8%) in an average period of 74.8 months. Combined therapy lead to favorable and durable results in perianal fistulizing Crohn's disease.

Variant Rh Alleles And Rh Immunisation In Patients With Sickle Cell Disease.

Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corresponding Rh antigen are considered autoantibodies in many cases but variant RH alleles found in SCD patients can also contribute to Rh alloimmunisation. In this study, we characterised variant RH alleles in 31 SCD patients who made antibodies to Rh antigens despite antigen-positive status and evaluated the clinical significance of the antibodies produced. RHD and RHCE BeadChip™ from BioArray Solutions and/or amplification and sequencing of exons were used to identify the RH variants. The serological features of all Rh antibodies in antigen-positive patients were analysed and the clinical significance of the antibodies was evaluated by retrospective analysis of the haemoglobin (Hb) levels before and after transfusion; the change from baseline pre-transfusion Hb and the percentage of HbS were also determined. We identified variant RH alleles in 31/48 (65%) of SCD patients with Rh antibodies. Molecular analyses revealed the presence of partial RHD alleles and variant RHCE alleles associated with altered C and e antigens. Five patients were compound heterozygotes for RHD and RHCE variants. Retrospective analysis showed that 42% of antibodies produced by the patients with RH variants were involved in delayed haemolytic transfusion reactions or decreased survival of transfused RBC. In this study, we found that Rh antibodies in SCD patients with RH variants can be clinically significant and, therefore, matching patients based on RH variants should be considered.

National Institutes Of Health Consensus Development Project On Criteria For Clinical Trials In Chronic Graft-versus-host Disease: I. The 2014 Diagnosis And Staging Working Group Report.

The 2005 National Institutes of Health (NIH) Consensus Conference proposed new criteria for diagnosing and scoring the severity of chronic graft-versus-host disease (GVHD). The 2014 NIH consensus maintains the framework of the prior consensus with further refinement based on new evidence. Revisions have been made to address areas of controversy or confusion, such as the overlap chronic GVHD subcategory and the distinction between active disease and past tissue damage. Diagnostic criteria for involvement of mouth, eyes, genitalia, and lungs have been revised. Categories of chronic GVHD should be defined in ways that indicate prognosis, guide treatment, and define eligibility for clinical trials. Revisions have been made to focus attention on the causes of organ-specific abnormalities. Attribution of organ-specific abnormalities to chronic GVHD has been addressed. This paradigm shift provides greater specificity and more accurately measures the global burden of disease attributed to GVHD, and it will facilitate biomarker association studies.

Post Hoc Analysis Of The Patricia Randomized Trial Of The Efficacy Of Human Papillomavirus Type 16 (hpv-16)/hpv-18 As04-adjuvanted Vaccine Against Incident And Persistent Infection With Nonvaccine Oncogenic Hpv Types Using An Alternative Multiplex Type-specific Pcr Assay For Hpv Dna.

The efficacy of the human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against cervical infections with HPV in the Papilloma Trial against Cancer in Young Adults (PATRICIA) was evaluated using a combination of the broad-spectrum L1-based SPF10 PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA25) system with type-specific PCRs for HPV-16 and -18. Broad-spectrum PCR assays may underestimate the presence of HPV genotypes present at relatively low concentrations in multiple infections, due to competition between genotypes. Therefore, samples were retrospectively reanalyzed using a testing algorithm incorporating the SPF10 PCR-DEIA/LiPA25 plus a novel E6-based multiplex type-specific PCR and reverse hybridization assay (MPTS12 RHA), which permits detection of a panel of nine oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 45, 52, 58, and 59). For the vaccine against HPV types 16 and 18, there was no major impact on estimates of vaccine efficacy (VE) for incident or 6-month or 12-month persistent infections when the MPTS12 RHA was included in the testing algorithm versus estimates with the protocol-specified algorithm. However, the alternative testing algorithm showed greater sensitivity than the protocol-specified algorithm for detection of some nonvaccine oncogenic HPV types. More cases were gained in the control group than in the vaccine group, leading to higher point estimates of VE for 6-month and 12-month persistent infections for the nonvaccine oncogenic types included in the MPTS12 RHA assay (types 31, 33, 35, 45, 52, 58, and 59). This post hoc analysis indicates that the per-protocol testing algorithm used in PATRICIA underestimated the VE against some nonvaccine oncogenic HPV types and that the choice of the HPV DNA testing methodology is important for the evaluation of VE in clinical trials. (This study has been registered at ClinicalTrials.gov under registration no. NCT00122681.).

Transcutaneous Tibial Nerve Stimulation In The Treatment Of Lower Urinary Tract Symptoms And Its Impact On Health-related Quality Of Life In Patients With Parkinson Disease: A Randomized Controlled Trial.

A randomized controlled trial study was performed to evaluate the efficacy of transcutaneous tibial nerve stimulation (TTNS) and sham TTNS, in patients with Parkinson disease (PD) with lower urinary tract symptoms (LUTS). Randomized controlled trial. Thirteen patients with a diagnosis of PD and bothersome LUTS were randomly allocated to one of the following groups: Group I: TTNS group (n = 8) and group II: Sham group (n = 5). Both groups attended twice a week during 5 weeks; each session lasted 30 minutes. Eight patients received TTNS treatment and 5 subjects allocated to group II were managed with sham surface electrodes that delivered no electrical stimulation. Assessments were performed before and after the treatment; they included a 3-day bladder diary, Overactive Bladder Questionnaire (OAB-V8), and the International Consultation on Incontinence Quality of Life Questionnaire Short Form (ICIQ-SF), and urodynamic evaluation. Following 5 weeks of treatment, patients allocated to TTNS demonstrated statistically significant reductions in the number of urgency episodes (P = .004) and reductions in nocturia episodes (P < .01). Participants allocated to active treatment also showed better results after treatment in the OAB-V8 and ICIQ-SF scores (P < .01, respectively). Urodynamic testing revealed that patients in the active treatment group showed improvements in intravesical volume at strong desire to void (P < .05) and volume at urgency (P < .01) when compared to subjects in the sham treatment group. These findings suggest that TTNS is effective in the treatment of LUTS in patients with PD, reducing urgency and nocturia episodes and improving urodynamic parameters as well as symptom scores measured by the OAB-V8 and health-related quality-of-life scores measured by the ICIQ-SF.

Bartonella Spp. Bacteremia In Blood Donors From Campinas, Brazil.

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.

Elevated Hypercoagulability Markers In Hemoglobin Sc Disease.

Hemoglobin SC disease is a very prevalent hemoglobinopathy, however very little is known specifically about this condition. There appears to be an increased risk of thromboembolic events in hemoglobin SC disease, but studies evaluating the hemostatic alterations are lacking. We describe a cross-sectional observational study evaluating coagulation activation markers in adult hemoglobin SC patients, in comparison with sickle cell anemia patients and healthy controls. A total of 56 hemoglobin SC and 39 sickle cell anemia patients were included in the study, all in steady state, and 27 healthy controls. None of the patients were in use of hydroxyurea. Hemoglobin SC patients presented a significantly up-regulated relative expression of tissue factor, as well as elevations in thrombin-antithrombin complex and D-dimer, in comparison to controls (p<0.01). Hemoglobin SC patients presented lower tissue factor expression, and thrombin-antithrombin complex and D-dimer levels when compared to sickle cell anemia patients (p<0.05). Endothelial activation (soluble thrombomodulin and soluble vascular cell adhesion molecule-1), and inflammation (tumor necrosis factor-alpha) markers were both significantly elevated in hemoglobin SC patients when compared to controls, being as high as the levels seen in sickle cell anemia. Overall, in hemoglobin SC patients, higher hemolytic activity and inflammation were associated with a more intense activation of coagulation, and hemostatic activation was associated with two very prevalent chronic complications seen in hemoglobin SC disease: retinopathy and osteonecrosis. In summary, our results demonstrate that hemoglobin SC patients present a hypercoagulable state, although this manifestation was not as intense as that seen in sickle cell anemia.

Long-term Efficacy And Safety Of Tenofovir Disoproxil Fumarate In Hiv-1-infected Adolescents Failing Antiretroviral Therapy: The Final Results Of Study Gs-us-104-0321.

Reports of long-term tenofovir disoproxil fumarate (TDF) treatment in HIV-infected adolescents are limited. We present final results from the open-label (OL) TDF extension following the randomized, placebo (PBO)-controlled, double-blind phase of GS-US-104-0321 (Study 321). HIV-infected 12- to 17-year-olds treated with TDF 300 mg or PBO with an optimized background regimen (OBR) for 24-48 weeks subsequently received OL TDF plus OBR in a single arm study extension. HIV-1 RNA and safety, including bone mineral density (BMD), was assessed in all TDF recipients. Eighty-one subjects received TDF (median duration 96 weeks). No subject died or discontinued OL TDF for safety/tolerability. At week 144, proportions with HIV-1 RNA <50 copies/mL were 30.4% (7 of 23 subjects with baseline HIV-1 RNA >1000 c/mL initially randomized to TDF), 41.7% (5 of 12 subjects with HIV-1 RNA <1000 c/mL who switched PBO to TDF) and 0% (0 of 2 subjects failed randomized PBO plus OBR with HIV-1 RNA >1000 c/mL and switched PBO to TDF). Viral resistance to TDF occurred in 1 subject. At week 144, median decrease in estimated glomerular filtration rate was 38.1 mL/min/1.73 m (n = 25). Increases in median spine (+12.70%, n = 26) and total body less head BMD (+4.32%, n = 26) and height-age adjusted Z-scores (n = 21; +0.457 for spine, +0.152 for total body less head) were observed at week 144. Five of 81 subjects (6%) had persistent >4% BMD decreases from baseline. Some subjects had virologic responses to TDF plus OBR, and TDF resistance was rare. TDF was well tolerated and can be considered for treatment of HIV-infected adolescents.

Low Bone Mass In Human Immunodeficiency Virus-infected Climacteric Women Receiving Antiretroviral Therapy: Prevalence And Associated Factors.

Low bone mineral density (BMD) has been found in human immunodeficiency virus (HIV)-infected patients; however, data on associated factors remain unclear, specifically in middle-aged women. This study aims to evaluate factors associated with low BMD in HIV-positive women. In this cross-sectional study, a questionnaire was administered to 206 HIV-positive women aged 40 to 60 years who were receiving outpatient care. Clinical features, laboratory test results, and BMD were assessed. Yates and Pearson χ(2) tests and Poisson multiple regression analysis were performed. The median age of women was 47.7 years; 75% had nadir CD4 T-cell counts higher than 200, and 77.8% had viral loads below the detection limit. There was no association between low BMD at the proximal femur and lumbar spine (L1-L4) and risk factors associated with HIV infection and highly active antiretroviral therapy. Poisson multiple regression analysis showed that the only factor associated with low BMD at the proximal femur and lumbar spine was postmenopause status. Low BMD is present in more than one third of this population sample, in which most women are using highly active antiretroviral therapy and have a well-controlled disease. The main associated factor is related to estrogen deprivation. The present data support periodic BMD assessments in HIV-infected patients and highlight the need to implement comprehensive menopausal care for these women to prevent bone loss.

Obstacle Crossing With Dual Tasking Is A Danger For Individuals With Alzheimer's Disease And For Healthy Older People.

The aim of this study was to analyze the effects of dual tasking on obstacle crossing during walking by individuals with Alzheimer's disease (AD) and by healthy older people. Thirty four elderly individuals (16 healthy subjects and 18 individuals with AD) were recruited to participate in this study. Three AD individuals and one control participant were excluded due to exclusion criteria. The participants were instructed to walk barefoot at their own speed along an 8 m long pathway. Each participant performed five trials for each condition (unobstructed walking, unobstructed walking with dual tasking, and obstacle crossing during walking with dual tasking). The trials were completely randomized for each participant. The mid-pathway stride was measured in the unobstructed walking trials and the stride that occurred during the obstacle avoidance was measured in the trials that involved obstacle crossing. The behavior of the healthy elderly subjects and individuals with AD was similar for obstacle crossing during walking with dual tasking. Both groups used the posture first strategy to prioritize stability and showed decreased attention to executive tasking while walking. Additionally, AD had a strong influence on the modifications that are made by the elderly while walking under different walking conditions.