Usefulness of vaginal cytology tests in women with previous hysterectomy for benign diseases: assessment of 53,891 tests

Objective. To assess the value of vaginal screening cytology after hysterectomy for benign disease.Methods. This cross-sectional study used cytology audit data from 2,512,039 screening tests in the metropolitan region of Campinas from 2000 to 2012; the object was to compare the prevalence of abnormal tests in women who had undergone a hysterectomy for benign diseases (n = 53,891) to that of women who had had no hysterectomy. Prevalence ratios (95% confidence intervals, 95% Cl) were determined, and chi-square analysis, modified by the Cochrane-Armitage test for trend, was used to investigate the effects of age.Results. The prevalence of atypical squamous cells (ASC), low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion or squamous-cell carcinoma (HSIL/SCC) was 0.13%, 0.04% and 0.03%, respectively, in women who had undergone hysterectomy, and 0.93%, 0.51% and 0.26% in women who had not undergone hysterectomy. The prevalence ratios for ASC, LSIL and HSIL/SCC were 0.14(0.11-0.17), 0.08 (0.06-0.13) and 0.13 (0.08-020), respectively, in women with a hysterectomy versus those without. For HSIL/SCC, the prevalence ratios were 0.09 and 029, respectively, for women <50 or >= 50 years. The prevalence rates in women with a previous hysterectomy showed no significant variation with age.Conclusion. The prevalence rates of ASC, LSIL and HSIL/SCC were significantly lower in women with a previous hysterectomy for benign disease compared with those observed in women with an intact uterine cervix. This study reinforces the view that there is no evidence that cytological screening is beneficial for women who have had a hysterectomy for benign disease. (C) 2015 Elsevier Inc. All rights reserved.

NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B4 in a murine model of gout

Objective Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)derived leukotriene B4 (LTB4) in driving tissue inflammation and hypernociception in a murine model of gout. Methods. Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1 beta (IL-1 beta), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB4 activity, cytokine (IL-1 beta, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. Results. Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophildependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1 beta/MyD88-dependent manner. LTB4 was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1 beta production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB4 after MSU crystal injection, and LTB4 was relevant in the MSU crystalinduced maturation of IL-1 beta. Mechanistically, LTB4 drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. Conclusion. These results reveal the role of the NLRP3 inflammasome in mediating MSU crystalinduced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB4 in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

ACUTE EFFECTS OF A WARM-UP INCLUDING ACTIVE, PASSIVE, AND DYNAMIC STRETCHING ON VERTICAL JUMP PERFORMANCE

Carvalho, FLP, Carvalho, MCGA, Simao, R, Gomes, TM, Costa, PB, Neto, LB, Carvalho, RLP, and Dantas, EHM. Acute effects of a warm-up including active, passive, and dynamic stretching on vertical jump performance. J Strength Cond Res 26(9): 2447-2452, 2012-The purpose of this study was to examine the acute effects of 3 different stretching methods combined with a warm-up protocol on vertical jump performance. Sixteen young tennis players (14.5 +/- 2.8 years; 175 +/- 5.6 cm; 64.0 +/- 11.1 kg) were randomly assigned to 4 different experimental conditions on 4 successive days. Each session consisted of a general and specific warm-up, with 5 minutes of running followed by 10 jumps, accompanied by one of the subsequent conditions: (a) Control Condition (CC)-5 minutes of passive rest; (b) Passive Stretching Condition (PSC)-5 minutes of passive static stretching; (c) Active Stretching Condition (ASC)-5 minutes of active static stretching; and (d) Dynamic Stretching Condition (DC)-5 minutes of dynamic stretching. After each intervention, the subjects performed 3 squat jumps (SJs) and 3 countermovement jumps (CMJs), which were measured electronically. For the SJ, 1-way repeated measures analysis of variance (CC x PSC x ASC x DC) revealed significant decreases for ASC (28.7 +/- 4.7 cm; p = 0.01) and PSC (28.7 +/- 4.3 cm; p = 0.02) conditions when compared with CC (29.9 +/- 5.0 cm). For CMJs, there were no significant decreases (p > 0.05) when all stretching conditions were compared with the CC. Significant increases in SJ performance were observed when comparing the DC (29.6 +/- 4.9 cm; p = 0.02) with PSC (28.7 +/- 4.3 cm). Significant increases in CMJ performance were observed when comparing the conditions ASC (34.0 +/- 6.0 cm; p = 0.04) and DC (33.7 +/- 5.5 cm; p = 0.03) with PSC (32.6 +/- 5.5 cm). A dynamic stretching intervention appears to be more suitable for use as part of a warm-up in young athletes.

Adipose Tissue-Derived Stem Cell Treatment Prevents Renal Disease Progression

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 x 10(5) ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 x 10(5) ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4. IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-alpha, KC, RANTES, and IL-1 alpha. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: An EEG wavelet analysis and retrograde neuronal tracer approach

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG + projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background. (C) 2012 Elsevier Inc. All rights reserved.

IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120 d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120 d a sustained production of cytokines (KC, IL-5, TNF-alpha, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(Pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A. (C) 2012 Elsevier Ltd. All rights reserved.

Aprendizado eletrônico na formação multiprofissional em saúde: avaliação inicial

O objetivo deste relato é apresentar, dentro da proposta do Programa Nacional de Reorientação da Formação Profissional em Saúde (Pró-Saúde), uma parte do projeto da Faculdade de Medicina de Ribeirão Preto (USP). Trata-se da estratégia de inserção das Tecnologias de Informação e Comunicação (TIC) no ensino de graduação extramuros da FMRP, que visa definir e implantar recursos tecnológicos de Aprendizado Eletrônico para apoiar atividades discentes e docentes, gestão da informação, educação continuada e segunda opinião formativa. A trajetória metodológica delineou tanto o processo de atendimento de parte das ações do eixo Cenário de Prática em Atenção Básica de saúde relativa ao processo de ampliação da rede de malha ótica, essencial para suporte às atividades de desenvolvimento do uso das TIC, quanto a abordagem qualitativa de um estudo exploratório sobre a utilização do Teleduc no primeiro ano do eixo de Atenção à Saúde da Comunidade (ASC) do curso de Medicina. Nesta investigação foram realizados dois grupos focais com aplicação de questionário estruturado a discentes e docentes.

NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy

Adenine overload promotes intratubular crystal precipitation and interstitial nephritis. We showed recently that these abnormalities are strongly attenuated in mice knockout for Toll-like receptors-2, -4, MyD88, ASC, or caspase-1. We now investigated whether NF-κB activation also plays a pathogenic role in this model. Adult male Munich-Wistar rats were distributed among three groups: C (n = 17), receiving standard chow; ADE (n = 17), given adenine in the chow at 0.7% for 1 wk and 0.5% for 2 wk; and ADE + pyrrolidine dithiocarbamate (PDTC; n = 14), receiving adenine as above and the NF-κB inhibitor PDTC (120 mg•kg-1•day-1 in the drinking water). After 3 wk, widespread crystal deposition was seen in tubular lumina and in the renal interstitium, along with granuloma formation, collagen accumulation, intense tubulointerstitial proliferation, and increased interstitial expression of inflammatory mediators. Part of the crystals were segregated from tubular lumina by a newly formed cell layer and, at more advanced stages, appeared to be extruded to the interstitium. p65 nuclear translocation and IKK-α increased abundance indicated activation of the NF-κB system. PDTC treatment prevented p65 migration and normalized IKK-α, limited crystal shift to the interstitium, and strongly attenuated interstitial fibrosis/inflammation. These findings indicate that the complex inflammatory phenomena associated with this model depend, at least in part, on NF-κB activation, and suggest that the NF-κB system may become a therapeutic target in the treatment of chronic kidney disease.

Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and -independent macrophage responses

NAIP5/NLRC4 (neuronal apoptosis inhibitory protein 5/nucleotide oligomerization domain-like receptor family, caspase activation recruitment domain domain-containing 4) inflammasome activation by cytosolic flagellin results in caspase-1-mediated processing and secretion of IL-1β/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC, and caspase-1 are required for IL-1β secretion in response to cytosolic flagellin, cell death, nevertheless, occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death is accompanied by IL-1α secretion and is temporally correlated with the restriction of Salmonella Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death do not require caspase activation but is regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributes to NAIP5/NLRC4 inflammasome-induced pyroptosis and IL-1α and IL-1β production in response to cytosolic flagellin. Together, our data describe a pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages

Mesenchymal stromal cell: new applications for regenerative medicine

In the last decades mesenchymal stromal cells (MSC), intriguing for their multilineage plasticity and their proliferation activity in vitro, have been intensively studied for innovative therapeutic applications. In the first project, a new method to expand in vitro adipose derived-MSC (ASC) while maintaining their progenitor properties have been investigated. ASC are cultured in the same flask for 28 days in order to allow cell-extracellular matrix and cell-cell interactions and to mimic in vivo niche. ASC cultured with this method (Unpass cells) were compared with ASC cultured under classic condition (Pass cells). Unpass and Pass cells were characterized in terms of clonogenicity, proliferation, stemness gene expression, differentiation in vitro and in vivo and results obtained showed that Unpass cells preserve their stemness and phenotypic properties suggesting a fundamental role of the niche in the maintenance of ASC progenitor features. Our data suggests alternative culture conditions for the expansion of ASC ex vivo which could increase the performance of ASC in regenerative applications. In vivo MSC tracking is essential in order to assess their homing and migration. Super-paramagnetic iron oxide nanoparticles (SPION) have been used to track MSC in vivo due to their biocompatibility and traceability by MRI. In the second project a new generation of magnetic nanoparticles (MNP) used to label MSC were tested. These MNP have been functionalized with hyperbranched poly(epsilon-lysine)dendrons (G3CB) in order to interact with membrane glycocalix of the cells avoiding their internalization and preventing any cytotoxic effects. In literature it is reported that labeling of MSC with SPION takes long time of incubation. In our experiments after 15min of incubation with G3CB-MNP more then 80% of MSC were labeled. The data obtained from cytotoxic, proliferation and differentiation assay showed that labeling does not affect MSC properties suggesting a potential application of G3CB nano-particles in regenerative medicine.

Functional dna nanostructures for in vitro biosensing in live cells

DNA is a fascinating biomolecule that is well known for its genetic role in living systems. The emerging area of DNA nanotechnology provides an alternative view that exploits unparallel self-assembly ability of DNA molecules for material use of DNA. Although many reports exist on the results of DNA self-assembling systems, still few of them focus on the in vitro study about the function of such DNA nanostructures in live cells. Due to this, there are still a limited research about the in vitro functionality of such designs. To address an aspect of this issue, we have designed, synthesized and characterized two multifunctional fluorescencent nanobiosensors by DNA self-assembling. Each structure was designed and implemented to be introduced in live cells in order to give information on their functioning in real-time. Computational tools were used in order to design a graphic model of two new DNA motifs and also to obtain the specific sequences to all the ssDNA molecules. By thermal self-assembly techniques we have successfully synthesized the structure and corroborate their formation by the PAGE technique. In addition, we have established the conditions to characterize their structural conformation change when they perform their sensor response. The sensing behavior was also accomplished by fluorescence spectroscopy techniques; FRET evaluation and fluorescence microscopy imaging. Providing the evidence about their adequate sensing performance outside and inside the cells detected in real-time. In a preliminary evaluation we have tried to show the in vitro functionality of our structures in different cancer cell lines with the ability to perform local sensing responses. Our findings suggest that DNA sensor nanostructures could serve as a platform to exploit further therapeutic achievements in live cells.

Pre-edge analysis of X-rays absorption spectra in TiO2 modified films

The thesis is mainly focused on the pre-edge analysis of XAS spectra of Ti HCF sample hexacyanocobaltate and hexacyanoferrate samples doped on a Indium Tin Oxide (ITO) thin film. The work is aimed at the determination of Ti oxidation state, as well as indication of various coordination number in the studied samples. The experiment have been conducted using XAFS (X-ray absorption fine structure)beamline at Elettra synchrotron, Trieste (Italy) under supervision of Professor Marco Giorgetti, Department of Industrial Chemistry, University of Bologna. The Master thesis accreditation to fullfill the ASC Master of Advanced Spectroscopy in Chemistry Degree requirement.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.