HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex.

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

Genetic deletion of Nox4 enhances cancerogen-induced formation of solid tumors.

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of H2O2 By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of H2O2 maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.

NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44-/- mice but not Cd14-/- mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4.

AIM: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. RESULTS: TGFß1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. INNOVATION & CONCLUSION: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.

NoxO1 Controls Proliferation of Colon Epithelial Cells.

Aim: Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut. Results: NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells. Conclusion: NoxO1 affects colon epithelium homeostasis and prevents inflammation.

Detection of Hydrogen Peroxide with Fluorescent Dyes.

SIGNIFICANCE: Hydrogen peroxide (H2O2) is a powerful effector of redox signaling. It is able to oxidize cysteine residues, metal ion centers, and lipids. Understanding H2O2-mediated signaling requires, to some extent, measurement of H2O2 level. Recent Advances: Chemically and genetically encoded fluorescent probes for the detection of H2O2 are currently the most sensitive and popular. Novel probes are constantly being developed, with the latest progress particular with boronates and genetically encoded probes. CRITICAL ISSUES: All currently available probes display limitations in terms of sensitivity, local and temporal resolution, and specificity in the detection of low H2O2 concentrations. In this review, we discuss the power of fluorescent probes and the systems in which they have been successfully employed. Moreover, we recommend approaches for overcoming probe limitations and for the avoidance of artifacts. FUTURE DIRECTIONS: Constant improvements will lead to the generation of probes that are not only more sensitive but also specifically tailored to individual cellular compartments. Antioxid. Redox Signal. 29, 585-602.

Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays.

Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR-/- abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH-dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. IN CONCLUSION: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays.

Knock out of the NADPH oxidase Nox4 has no impact on life span in mice.

The free radical theory of aging suggests reactive oxygen species as a main reason for accumulation of damage events eventually leading to aging. Nox4, a member of the family of NADPH oxidases constitutively produces ROS and therefore has the potential to be a main driver of aging. Herein we analyzed the life span of Nox4 deficient mice and found no difference when compared to their wildtype littermates. Accordingly neither Tert expression nor telomere length was different in cells isolated from those animals. In fact, Nox4 mRNA expression in lungs of wildtype mice dropped with age. We conclude that Nox4 has no influence on lifespan of healthy mice.

Aplicação de ácido poli-l-lático para o tratamento da flacidez corporal / Poly-L-lactic acid injections in sagging body skin

Introdução: O tratamento da flacidez cutânea corporal constitui grande desafio, pois poucos são os procedimentos destinados a melhorá-la. O ácido poli-L-lático é polímero sintético injetável da família dos alfa-hidroxiácidos, cuja injeção na derme profunda ou hipoderme superficial induz reação local e gradual, com síntese de novo colágeno pelos fibroblastos e consequente aumento da espessura dérmica. Objetivo: Avaliar os efeitos do ácido poli-L-lático na flacidez da pele do corpo. Métodos: Quatorze pacientes que apresentavam flacidez de região glútea foram tratadas com duas aplicações de ácido poli-L-lático com intervalos de 45 dias entre elas. Foram realizadas avalição clínica inicial e seis meses após a segunda aplicação, fotos prévias e seis meses após o tratamento, bem como foram aplicados questionários às pacientes e ao médico avaliador, e realizado exame ultrassonográfico de todas as pacientes para verificação do aumento de espessura dérmica. Foram avaliados também os efeitos adversos observados. Resultados: Na opinião das pacientes, 85% apresentaram melhora no aspecto geral da pele e 71% na flacidez. Para o médico avaliador, 100% das pacientes apresentaram melhora no aspecto geral da pele e na flacidez, porém em graus variáveis e não proporcionais ao grau de gravidade inicial. Onze pacientes tiveram aumento da espessura dérmica acima de 20% dos pontos. Não houve efeitos colaterais importantes no período avaliado. Conclusões: Os resultados são promissores e devem ser confirmados com a realização de estudos com casuística maior.

Introduction: The treatment of sagging skin in body areas is still a big challenge, as there are few aesthetic procedures aiming to improve it. The poly-L-lactic acid (PLLA) is an injectable synthetic polymer of the alpha-hydroxy acids family, which injection into the deep dermis or superficial hypodermis induces a local and gradual reaction, with synthesis of new collagen by the fibroblasts and consequent increase of dermal thickness. Purpose: Evaluate the effects of poly-L-lactic acid on the sagging skin of the body. Methods: Fourteen patients with sagging in gluteal region underwent two applications of poly-L-lactic acid with a 45 days interval between them. We performed an initial clinical evaluation and after six months of the second application, photos before and after six months of treatment, questionnaires answered by the patient and by the evaluating physician and ultrasound examination of all patients to assess the increase of dermal thickness. The adverse effects observed were also evaluated. Results: In patient's opinion, 85% presented improvement of general appearance of the skin and 71% noticed improvement of sagging. For the evaluating physician, 100% of patients presented improvement of general appearance of the skin and improvement of sagging, but in different degrees and not proportional to the initial degree of severity. There was no significant side effects in the period evaluated. Conclusions: The results are promising and should be confirmed with studies with a larger sample size.

The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype.

OBJECTIVE: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. APPROACH AND RESULTS: A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1(-/-) when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1(-/-) lung endothelial cells (LECs) and a more tip-cell-like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1(-/-) LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1(-/-) LECs when compared with wild-type LECs. CONCLUSIONS: NoxO1 stimulates α-secretase activity probably through reactive oxygen species-mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

Unchanged NADPH Oxidase Activity in Nox1-Nox2-Nox4 Triple Knockout Mice: What Do NADPH-Stimulated Chemiluminescence Assays Really Detect?

NADPH oxidases of the Nox family are considered important sources of cellular reactive oxygen species (ROS) production. This conclusion is, in part, based on the ability of NADPH to elicit a chemiluminescence signal in tissue/cell homogenates or membrane preparations in the presence of enhancers such as lucigenin, luminol, or L012. However, the ability of these particular assays to specifically detect Nox activity and Nox-derived ROS has not been proven. In this study, we demonstrate that combined knockout of the three main Nox enzymes of the mouse (Nox1-Nox2-Nox4 triple knockout) had no impact on NADPH-stimulated chemiluminescence signals in the aorta, heart, and kidney homogenates. In the NADPH-stimulated membrane assays, no effect of in vivo angiotensin II pretreatment or deletion of Nox enzymes was observed. In in vitro studies in HEK293 cells, the overexpression of Nox5 or Nox4 markedly increased ROS production in intact cells, whereas overexpression of Nox5 or Nox4 had no influence on the signal in membrane assays. In contrast, overexpression of nitric oxide synthase or cytochrome P450 enzymes resulted in an increased chemiluminescence signal in isolated membranes. On the basis of these observations, we propose the hypothesis that NADPH-stimulated chemiluminescence-based membrane assays, as currently used, do not reflect Nox activity.

Clinical presentation of pili torti - Case report

Pili torti also known as ‘twisted hairs’ (Latin: pili=hair; torti=twisted) is a rare, congenital or acquired clinical presentation, in which the hair shaft is flattened at irregular intervals and twisted 180º along its axis. It is clinically characterized by fragile, brittle, coarse and lusterless hairs, due to uneven light reflection on the twisted hair surface. Pili torti may be associated with neurological abnormalities and ectodermal dysplasias. There is no specifi c treatment for this condition, but it may improve spontaneously after puberty. We report a case of pili torti in a child who presented fragile, brittle, diffi cult to comb hair. The patient had no comorbidities.

Copper sulfate acute ecotoxicity and environmental risk for tropical fish / Ecotoxicidade aguda e risco ambiental do sultado de cobre para peixes tropicais

The aim of this study was to estimate copper sulfate acute toxicity and to determine death percentage and environmental risk on guppy fish (Phallocerus caudimaculatus), zebrafish (Brachydanio rerio), mato grosso (Hyphessobrycon eques), and pacu (Piaractus mesopotamicus). Fish were exposed to 0.01, 0.03, 0.05, 0.07, 0.10, and 0.30 mg L-1 (guppy), 0.05, 0.07, 0.10, and 0.30 mg L-1 (zebrafish), 0.07, 0.10, 0.20, and 0.30 mg L-1 (mato grosso) and 9.5, 10.0, 10.5, 11.0, 11.5, and 12.0 mg L-1 (pacu) of copper sulfate, with triplicate control. The estimated 50% average lethal concentrations (LC50; 96 hours) were 0.05 (guppy), 0.13 (zebrafish); 0.16 (mato grosso) and 10.36 mg L-1 (pacu). Copper sulfate was extremely toxic for guppy, highly toxic for zebrafish and mato grosso and lightly toxic for pacu and presents environmental risk of high adverse effects on the guppy, zebrafish and mato grosso and moderate adverse effect to the pacu. Therefore, the guppy fish, zebrafish, and mato grosso are important alternatives for copper sulfate toxicity evaluation in waterbodies.

O objetivo deste estudo foi estimar a toxicidade aguda, determinar a porcentagem de mortalidade e o risco ambiental do sulfato de cobre para o peixe guaru (Phallocerus caudimaculatus), zebrafish (Brachydanio rerio), mato grosso (Hyphessobryconeques) e pacu (Piaractus mesopotamicus). Para realização dos ensaios com sulfato de cobre foram utilizados: 0,01; 0,03; 0,05; 0,07; 0,10 e 0,30 mg L-1 (guaru), 0,05; 0,07; 0,10 e 0,30 mg L-1 (paulistinha), 0,07; 0,10; 0,20 e 0,30 mg L-1 (mato grosso) e 9,5; 10,0; 10,5; 11,0; 11,5 e 12,0 mg L-1 (pacu) como controle em triplicata. A concentração letal média 50% (CL50; 96h) estimada para o guaru foi de 0,05, para o paulistinha foi de 0,13 para o mato grosso foi de 0,16 e para o pacu foi de 10,36 mg L-1. O sulfato de cobre foi classificado como extremamente tóxico para o guaru, altamente tóxico para o paulistinha e mato grosso e ligeiramente tóxico para o pacu e apresenta risco ambiental de elevado efeito adverso para o guaru, paulistinha e mato grosso e moderado efeito adverso para o pacu. Assim, conclui-se que o guaru, paulistinha e mato grosso são importantes alternativas para avaliação da toxicidade do sulfato de cobre em corpos hídricos.

Redox-relevant aspects of the extracellular matrix and its cellular contacts via integrins.

SIGNIFICANCE: The extracellular matrix (ECM) fulfills essential functions in multicellular organisms. It provides the mechanical scaffold and environmental cues to cells. Upon cell attachment, the ECM signals into the cells. In this process, reactive oxygen species (ROS) are physiologically used as signalizing molecules. RECENT ADVANCES: ECM attachment influences the ROS-production of cells. In turn, ROS affect the production, assembly and turnover of the ECM during wound healing and matrix remodeling. Pathological changes of ROS levels lead to excess ECM production and increased tissue contraction in fibrotic disorders and desmoplastic tumors. Integrins are cell adhesion molecules which mediate cell adhesion and force transmission between cells and the ECM. They have been identified as a target of redox-regulation by ROS. Cysteine-based redox-modifications, together with structural data, highlighted particular regions within integrin heterodimers that may be subject to redox-dependent conformational changes along with an alteration of integrin binding activity. CRITICAL ISSUES: In a molecular model, a long-range disulfide-bridge within the integrin ß-subunit and disulfide bridges within the genu and calf-2 domains of the integrin α-subunit may control the transition between the bent/inactive and upright/active conformation of the integrin ectodomain. These thiol-based intramolecular cross-linkages occur in the stalk domain of both integrin subunits, whereas the ligand-binding integrin headpiece is apparently unaffected by redox-regulation. FUTURE DIRECTIONS: Redox-regulation of the integrin activation state may explain the effect of ROS in physiological processes. A deeper understanding of the underlying mechanism may open new prospects for the treatment of fibrotic disorders.

Reação reversa macular da hanseníase: relato de caso / Macular reversal reaction in leprosy: a case report

A reação reversa maculosa consiste no aparecimento abrupto de máculas hipocrômicas, ocorrendo em pacientes hansenianos dimorfos que completaram o tratamento com poliquimioterapia para hanseníase multibacilar. Em geral, surgem entre 6 a 12 meses da alta, com baciloscopia negativa e boa resposta a corticoterapia sistêmica. Ressaltamos a dificuldade em diferenciar recidiva de um episódio reacional, já que não existem critérios clínicos bem estabelecidos que possibilitem este diagnóstico, além de existirem poucos relatos em literatura. Relatamos um caso clínico com diagnóstico de reação reversa macular após período variável de alta do tratamento de hanseniase dimorfa-dimorfa. Foi feita investigação por meio de anamnese rigorosa, exame dermatológico, exame histopatológico da lesão e baciloscopia, excluindo-se os critérios de recidiva, além de analisados dados anteriores do prontuário.O paciente foi submetido a corticoterapia sistêmica,apresentando melhora das lesões. Conclui-seque a reação reversa maculosa deve ser lembrada nos diagnósticos diferenciais com hanseníase recidivada e episódios reacionais clássicos, evitando retratamentos desnecessários.

Macular reversal reaction is the abrupt onset of hypochromic lesions, occurring in borderline leprosy patients who completed treatment with multidrugtherapy for multibacillary leprosy. In general, these reactions appear 6 to 12 months after medical discharge, showing negative skin smear and good response to systemic corticosteroid therapy. We emphasize the difficulty in differentiating relapse cases from leprosy reactions, as there are no well-established clinical criteria that allow this diagnosis, and moreover there are few reports about it in the literature. We report a borderline leprosy case diagnosed with macular reversal reaction after variable period of discharge from treatment. Detailed anamnesis, dermatological and histopathological examination and bacilloscopy, analysis of previous medical records, excluding the relapse criteria, were used for the investigation. The patient was submitted to systemic corticosteroid therapy, with improvement of the lesions. It is concluded that macular reversal reaction should be considered in the differential diagnosis of relapsed leprosy and classic reactional episodes, avoiding unnecessary retreatment.

Retorno ao trabalho de pacientes com amputação traumática de membros inferiores / Return to work in traumatic lower limb amputees patients

O paciente com amputação traumática é, em geral, aquele com pouca ou nenhuma comorbidade e está no auge da vida produtiva. Mesmo em condições de reabilitação adequada, diversos autores têm citado dificuldade no processo de retorno à atividade laborativa, e a sua relação com outros determinantes além da aptidão física. Objetivo: Avaliamos o índice de retorno ao trabalho após reabilitação de pacientes atendidos no Lar Escolar São Francisco de Jan/2007 a Dez/2010 com amputação traumática de membros inferiores. Método: Foram pesquisados os fatores sociais e econômicos possivelmente relacionados a esse desfecho. A amostra final foi de 13 pacientes, todos com amputação unilateral, com uso regular da prótese. Dois eram do sexo feminino. Nove (69%) retornaram ao trabalho. Outras seqüelas consideráveis estavam presentes em 23% dos pacientes - lesão de plexo braquial e dor fantasma - e se mostrou o fator isolado mais importante para o não retorno ao trabalho. Resultados: Não encontramos relação importante entre retorno ao trabalho e fatores como recebimento de benefício previdenciário, idade ou amputação por acidente de trabalho. Conclusão: Há dados ainda inconclusivos que justificam a realização de novos estudos sobre a relação independente entre os diversos fatores mencionados e o retorno ao trabalho de pacientes amputados.

Generally, traumatic amputee patients have little or no comorbidities, and they are at the apogee of productive life. Even in good rehabilitation program conditions, the literature points to difficulties in the return-to-work process, and its relationship with other determinants besides physical aptitude. Objective: We studied the rate of return-to-work after the rehabilitation process at Lar Escola São Francisco between January of 2007 and December of 2010 of patients that had undergone traumatic lower limb amputation. Method: Social and economic factors were studied that might have been related to returning to work. The final sample was 13 patients; all of them were unilateral amputees, who used prostheses regularly. Two of them were female. Nine of them (69%) returned to work. In 23% of the cases, other important sequelae were present - brachial plexus injuries and phantom pain - which appeared as the most important single factor for not returning to work. Results: We found no important relationship between returning to work and other factors such as paid benefits, age, or amputation due to work accident. Conclusion: The data is still inconclusive, which calls for more studies on the independent relationship between the various factors mentioned and amputee patients returning to work.

Prevalência de dor fantasma em amputados do Lar Escola São Francisco / Prevalence of phantom pain among amputees at Lar Escola São Francisco

A fisiopatologia da sensação dolorosa do membro fantasma, ou da dor fantasma, é caracterizada pela reorganização do mapeamento das estruturas representadas no córtex cerebral, um processo de plasticidade sensitiva e motora. Sua presença pode interferir na reabilitação física e psicossocial do paciente amputado, comprometendo a aquisição de habilidades e a qualidade de vida. Objetivo: Aferir a prevalência de dor fantasma nos pacientes amputados do Lar Escola São Francisco (LESF). Método: Foram pesquisados os prontuários dos pacientes atendidos no Grupo de Amputações e Próteses no período de janeiro de 2005 a dezembro de 2010 e analisada a presença ou ausência de queixa de dor fantasma. Resultados: Dos 330 prontuários analisados, em dez havia referencia de queixa de dor fantasma, equivalendo à proporção de 3,3% dos pacientes. Conclusão: A prevalência de dor fantasma foi baixa entre os amputados estudados do LESF. Sua abordagem precisa ser mais bem estudada dentro da avaliação individual do paciente amputado.

The physiopathology of phantom limb pain is characterized by cortical map reorganization, a process that involves plasticity in sensorimotor representations. The presence of phantom limb pain can interfere with the physical and psychosocial rehabilitation of amputees, compromising the patient?s acquisition of skills and quality of life. Objective: To determine the prevalence of phantom limb pain in amputees seen at the Lar Escola São Francisco (LESF). Methods: The records of patients attending the Amputation and Prosthesis Group between January 2005 and December 2010 were analyzed regarding the presence or absence of signs of phantom limb pain. Results: Phantom limb pain was reported in 10 of 330 records analyzed, corresponding to a proportion of 3.03%. Conclusion: Prevalence of phantom limb pain was low among amputees studied at LESF. Its approach needs to be better investigated during individual assessment of amputees.

Integrin α7ß1 is a redox-regulated target of hydrogen peroxide in vascular smooth muscle cell adhesion.

Upon adhesion to laminin-111, aortic smooth muscle cells initially form membrane protrusions with an average diameter of 2.9µm. We identified these protrusions also as subcellular areas of increased redox potential and protein oxidation by detecting cysteine sulfenic acid groups with dimedone. Hence, we termed these areas oxidative hot spots. They are spatially and temporally transient during an early stage of adhesion and depend on the activity of the H(2)O(2)-generating NADPH oxidase 4. Presumably located on cellular protrusions, integrin α7ß1 mediates adhesion and migration of vascular smooth muscle cells to laminins of their surrounding basement membrane. Using protein chemistry and mass spectrometry, two specific oxidation sites within the integrin α7 subunit were identified: one located in its genu region and another within its calf 2 domain. Upon H(2)O(2) treatment, two cysteine residues are oxidized thereby unlocking a disulfide bridge. The genu region is a hinge, around which the integrin domains pivot between a bent/inactive and an upright/active conformation. Also, cysteine oxidation within the calf 2 domain permits conformational changes related to integrin activation. H(2)O(2) treatment of α7ß1 integrin in concentrations of up to 100µM increases integrin binding activity to laminin-111, suggesting a physiological redox regulation of α7ß1 integrin.

Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom.

BACKGROUND: Multifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms. METHODS: The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity. RESULTS: Bl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H2O2. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC(50) of 0.07µM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H2O2 in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase. CONCLUSION: Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H2O2 which kill the cells. GENERAL SIGNIFICANCE: These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.