Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions

The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long-term-infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. the extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro-irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non-irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Escola Paulista Medi, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Trop Med, Lab Soroepidemiol & Imunobiol, São Paulo, BrazilFdn Oswaldo Cruz FIOCRUZ, INCT DT, Salvador, BrazilUniversidade Federal de São Paulo, Fac Med, Dept Prevent Med, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Medi, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Trop Med, Lab Soroepidemiol & Imunobiol, São Paulo, BrazilUniversidade Federal de São Paulo, Fac Med, Dept Prevent Med, São Paulo, BrazilFAPESP: 10/19335-4Web of Scienc

Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv)

<p>Abstract</p> <p>Background</p> <p>Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic <it>Escherichia coli </it>(EPEC) and enterohemorrhagic <it>Escherichia coli </it>(EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int_388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).</p> <p>Findings</p> <p>Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into <it>pGEM-T Easy </it>vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. <it>E. coli </it>BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69.</p> <p>Conclusion</p> <p>This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int_388-667) in purified form and the EPEC isolate.</p

Morphological changes of the caudal cervical intervertebral foramina due to flexion-extension and compression-traction movements in the canine cervical vertebral column

Abstract Background: Previous studies in humans have reported that the dimensions of the intervertebral foramina change significantly with movement of the spine. Cervical spondylomyelopathy (CSM) in dogs is characterized by dynamic and static compressions of the neural components, leading to variable degrees of neurologic deficits and neck pain. Studies suggest that intervertebral foraminal stenosis has implications in the pathogenesis of CSM. The dimensions of the cervical intervertebral foramina may significantly change during neck movements. This could have implication in the pathogenesis of CSM and other diseases associated with radiculopathy such as intervertebral disc disease. The purpose of this study was to quantify the morphological changes in the intervertebral foramina of dogs during flexion, extension, traction, and compression of the canine cervical vertebral column. All vertebral columns were examined with magnetic resonance imaging prior to biomechanic testing. Eight normal vertebral columns were placed in Group 1 and eight vertebral columns with intervertebral disc degeneration or/and protrusion were assigned to Group 2. Molds of the left and right intervertebral foramina from C4-5, C5-6 and C6-7 were taken during all positions and loading modes. Molds were frozen and vertical (height) and horizontal (width) dimensions of the foramina were measured. Comparisons were made between neutral to flexion and extension, flexion to extension, and traction to compression in neutral position. Results: Extension decreased all the foraminal dimensions significantly, whereas flexion increased all the foraminal dimensions significantly. Compression decreased all the foraminal dimensions significantly, and traction increased the foraminal height, but did not significantly change the foraminal width. No differences in measurements were seen between groups. Conclusions: Our results show movement-related changes in the dimensions of the intervertebral foramina, with significant foraminal narrowing in extension and compression

Oxazole Dyes With Potential For Photoluminescence Bioprobes: A Two-Photon Absorption Study

In this work, six π-conjugated oxazole compounds dissolved in dichloromethane were characterized with linear and nonlinear optical measurements. Z-scan with femtosecond laser pulses was employed to determine the two-photon absorption (TPA) spectra. Other photophysical parameters, such as absorbance, solvatochromism, lifetime fluorescence, and fluorescence anisotropy, were evaluated with linear optical techniques. The experimental TPA cross section spectra were adjusted by the sum-over-states (SOS) model, by which important parameters such as transition dipole moments and broadening parameters were determined. To better understand the TPA spectra of the oxazole compounds, quantum-chemical calculations using the response function formalism and the density functional theory level of theory were performed. Using the results provided by the quantum-chemical calculations and the broadening parameters estimated through the application of the SOS model, the TPA spectra were simulated by the superposition (summation) of individual homogeneous Lorentzian absorption profiles

Safety and Immunogenicity of an In Vivo Muscle Electroporation Delivery System for DNA-hsp65 Tuberculosis Vaccine in Cynomolgus Monkeys

A Bacille Calmette–Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime–boost regimen, to help the induction of a stronger cellular immune response

Estudo de seguimento por dois anos de idosos residentes em São Paulo, Brasil: metodologia e resultados preliminares

INTRODUCTION: Previous cross-sectional studies have shown a high prevalence of chronic disease and disability among the elderly. Given Brazil s rapid aging process and the obvious consequences of the growing number of old people with chronic diseases and associated disabilities for the provision of health services, a need was felt for a study that would overcome the limitations of cross-sectional data and shed some light on the main factors determining whether a person will live longer and free of disabling diseases, the so-called successful aging. The methodology of the first follow-up study of elderly residents in Brazil is presented. METHOD: The profile of the initial cohort is compared with previous cross-sectional data and an in-depth analysis of nonresponse is carried out in order to assess the validity of future longitudinal analysis. The EPIDOSO ( Epidemiologia do Idoso ) Study conducted a two-year follow-up of 1,667 elderly people (65+), living in S. Paulo. The study consisted of two waves, each consisting of household, clinical, and biochemical surveys. RESULTS AND CONCLUSIONS: In general, the initial cohort showed a similar profile to previous cross-sectional samples in S. Paulo. There was a majority of women, mostly widows, living in multigenerational households, and a high prevalence of chronic illnesses, psychiatric disturbances, and physical disabilities. Despite all the difficulties inherent in follow-up studies, there was a fairly low rate of nonresponse to the household survey after two years, which did not actually affect the representation of the cohort at the final household assessment, making unbiased longitudinal analysis possible. Concerning the clinical and blood sampling surveys, the respondents tended to be younger and less disabled than the nonrespondents, limiting the use of the clinical and laboratory data to longitudinal analysis aimed at a healthier cohort. It is worth mentioning that gender, education, family support, and socioeconomic status were not important determinants of nonresponse, as is often the case.INTRODUÇÃO: Estudos transversais recentes mostraram alta prevalência de doenças crônicas e incapacidades físicas entre idosos. Considerando o rápido processo de envelhecimento do Brasil e as conseqüências que esse aumento de idosos com doenças crônicas e incapacidades associadas acarretará para o sistema de saúde, fazia-se necessário estudo que pudesse superar as limitações dos dados transversais, permitindo determinar quais os fatores determinantes de uma vida longa e livre de doenças incapacitantes, o chamado envelhecimento bem sucedido. É apresentada a metodologia do primeiro estudo epidemiológico longitudinal com idosos residentes na comunidade, no Brasil. MÉTODO: O perfil do cohorte inicial é comparado com dados de estudos anteriores a com o perfil dos não respondentes para avaliar a validade de análises longitudinais futuras.O projeto EPIDOSO (Epidemiologia do Idoso) seguiu por dois anos 1.667 idosos (65+), residentes em São Paulo. Consistiu de duas ondas, cada qual com três inquéritos: domiciliar, clínico e laboratorial. RESULTADOS E CONCLUSÕES: O perfil da população não diferiu de estudos anteriores, mostrando maioria de mulheres, viúvas, vivendo em domicílios multigeracionais, com uma alta prevalência de doenças crônicas, distúrbios psiquiátricos e incapacidades físicas. A despeito de todas as dificuldades inerentes a um estudo longitudinal, o grupo de não-respondentes ao segundo inquérito domiciliar não diferiu significativamente dos respondentes, assegurando análises longitudinais livres desse tipo de viés. Em relação aos inquéritos clínico e laboratorial, os não-respondentes mostraram-se mais velhos e mais incapacitados que os respondentes, limitando o uso dos dados clínicos e laboratoriais a análises pertinentes a uma cohorte mais jovem e saudável. Sexo, educação, apoio familiar e nível socioeconômico não influenciaram de forma significativa a taxa de não - resposta, ao contrário do que se costuma verificar.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Multiplicities of charged pions and unidentified charged hadrons from deep-inelastic scattering of muons off an isoscalar target

Multiplicities of charged pions and unidentified hadrons produced in deep-inelastic scattering were measured in bins of the Bjorken scaling variable $x$, the relative virtual-photon energy $y$ and the relative hadron energy $z$. Data were obtained by the COMPASS Collaboration using a 160 GeV muon beam and an isoscalar target ($^6$LiD). They cover the kinematic domain in the photon virtuality $Q^2$ > 1(GeV/c$)^2$, $0.004 < x < 0.4$, $0.2 < z < 0.85$ and $0.1 < y < 0.7$. In addition, a leading-order pQCD analysis was performed using the pion multiplicity results to extract quark fragmentation functions

Identification of Eschweilenol C in derivative of Terminalia fagifolia Mart. and green synthesis of bioactive and biocompatible silver nanoparticles

A green synthetic route was developed to prepare silver nanoparticles (AgNPs) in aqueous solution for biological applications. Eschweilenol C, a compound derivative ellagic acid was identified as the main constituent of the aqueous fraction of the ethanolic extract of Terminalia fagifolia Mart. by NMR analysis. In the green synthesis, the ethanolic extract of T. fagifolia and its aqueous fraction were used to promote silver reduction and nanoparticle stabilization. The synthesized AgNPs presented a spherical or polygonal morphology shape by TEM analysis and AgNPs showed high levels of antioxidant and considerable antibacterial and antifungal activities. Synthesized nanoparticles presented significant antioxidant activity by sequestration of DPPH and ABTS radicals, in addition to iron reduction (FRAP assay) and measurement of antioxidant capacity in ORAC units, in addition, AgNP synthesized with the aqueous fraction also demonstrated antioxidant potential in microglial cells. Gram-positive and Gram-negative bacteria were susceptible to growth inhibition by the nanoparticles, among which the AgNPs formed by the ethanolic extract was the most effective. The data obtained by AFM images suggested that AgNPs could lead to the lysis of bacteria and subsequent death. The antifungal assays showed high efficiency against yeasts and dermatophytes. This work represents the first description of antifungal activity by AgNPs against Fonsecaea pedrosoi, the etiologic agent of chromoblastomycosis. In relation to biocompatibility, the AgNPs induced lower haemolysis than AgNO3.We thank Herbert Kogler and Reinhard Wimmer for the identification of Eschweilenol C. The NMR laboratory at Aalborg University is supported by the Obel Family, SparNord and Carlsberg foundations.The authors are grateful to Carla Eiras (LIMAV/CT/UFPI) and to FCT and EU for financial support through project UID/QUI/50006/2013– POCI-01-0145-FEDER-007265 from COMPETE and projectNORTE-01-0145-FEDER-000011 from COMPETE. Thanks to Andreia Pinto for help with the TEM measurements at Instituto de Medicina Molecular (IMM). This work was supported by the Histology and Comparative Pathology Laboratory of the IMMinfo:eu-repo/semantics/publishedVersio