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Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.
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Leishmania braziliensis , Leishmania , Leishmaniose Cutânea , Fosforilcolina/análogos & derivados , Humanos , Antimoniato de Meglumina/uso terapêutico , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/tratamento farmacológicoRESUMO
Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.
Assuntos
Exossomos , Leishmania braziliensis , Leishmania donovani , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Interleucina-6/farmacologia , MacrófagosRESUMO
Heavy metals are the main pollutants present in aquatic environments and their presence in human organisms can lead to many different diseases. While many methods exist for analysis, colorimetric and electrochemistry are particularly attractive for on-site analysis and their integration on a single platform can improve multiplexed metals analysis. This report describes for the first time a "plug-and-play" (PnP) assembly for coupling a microfluidic paper-based device (µPAD) and a screen-printed electrochemical paper-based device (ePAD) using a vertical and reversible foldable mechanism for multiplexed detection of Fe, Ni, Cu, Zn, Cd and Pb in river water samples. The integration strategy was based on a reversible assembly, allowing the insertion of a pretreatment zone to minimize potential chemical interfering agents and providing a better control of the aspirated sample volume as well as to a lower sample evaporation rate. In comparison with lateral flow and electrochemical assays performed using independent devices, the integrated prototype proved that the reversible coupling mechanism does not interfere on the analytical performance (95% confidence interval). The limit of detection (LOD) values calculated for metals determined varied from 0.1 to 0.3 mg L-1 (colorimetric) and from 0.9 to 10.5 µg L-1 (electrochemical). When compared to other integrated devices based on horizontal designs, the use of a foldable coupling mechanism offered linear response in a lower concentration range and better LOD values for Fe, Ni and Cu. The proposed method successfully measured heavy metals in river water samples with concentrations ranging from 16 to 786 µg L-1, with recovery studies ranging from 76 to 121%. The new method also showed good correlation with conventional atomic absorption spectroscopic methods (95% significance level). Thus, the integration of µPADs and ePADs by a vertical folding mechanism was efficient for multiplexed heavy metal analysis and could be exploited for environmental monitoring.
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The cell wall of wild-type (WT) Mycobacterium tuberculosis (Mtb), an etiologic agent of tuberculosis (TB) and a Mtb strain disrupted in a 13-gene operon mce1 (Δmce1) varies by more than 400 lipid species. Here, we examined Mtb lipid-induced response in murine macrophage, as well as in human T-cell subpopulations in order to gain an insight into how changes in cell wall lipid composition may modulate host immune response. Relative to WT Mtb cell wall lipids, the non-polar lipid extracts from Δmce1 enhanced the mRNA expression of lipid-sense nuclear receptors TR4 and PPAR-γ and dampened the macrophage expression of genes encoding TNF-α, IL-6, and IL-1ß. Relative to untreated control, WT lipid-pre-stimulated macrophages from healthy individuals induced a higher level of CD4-CD8- double negative T-cells (DN T-cells) producing TNF-α. Conversely, compared to WT, stimulation with Δmce1 lipids induced higher mean fluorescence intensity (MFI) in IL-10-producing DN T cells. Mononuclear cells from TB patients stimulated with WT Mtb lipids induced an increased production of TNF-α by CD8+ lymphocytes. Taken together, these observations suggest that changes in mce1 operon expression during a course of infection may serve as a strategy by Mtb to evade the host pro-inflammatory responses.
Assuntos
Proteínas de Bactérias/genética , Parede Celular/imunologia , Ativação Linfocitária/imunologia , Ativação de Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Adolescente , Adulto , Animais , Parede Celular/química , Feminino , Humanos , Lipídeos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Óperon , Células RAW 264.7 , Linfócitos T/imunologia , Adulto JovemRESUMO
This study describes an inexpensive and nonconventional soft-embossing protocol to produce microfluidic devices in poly(methyl methacrylate) (PMMA). The desirable microfluidic structure was photo-patterned in a poly(vinyl acetate) (PVAc) film deposited on glass substrate to produce a low-relief master. Then, this template was used to generate a high-relief pattern in stiffened PDMS by increasing of curing agent /monomer ratio (1:5) followed by thermal aging in a laboratory oven (200°C for 24 h). The stiffened PDMS masters were used to replicate microfluidic devices in PMMA based on soft embossing at 220-230°C and thermal sealing at 140°C. Both embossing and sealing stages were performed by using binder clips. The proposed protocol has ensured the replication of microfluidic devices in PMMA with great fidelity (>94%). Examples of MCE devices, droplet generator devices and spot test array were successfully demonstrated. For testing MCE devices, a mixture containing inorganic cations was selected as model and the achieved analytical performance did not reveal significant difference from commercial PMMA devices. Water droplets were successfully generated in an oil phase at rate of ca. 60 droplets/min (fixing the continuous phase flow rate at 100 µL/h) with size of ca. 322 ± 6 µm. Glucose colorimetric assay was performed on spot test devices and good detectability level (5 µmol/L) was achieved. The obtained results for two artificial serum samples revealed good agreement with the certified concentrations. Based on the fabrication simplicity and great analytical performance, the proposed soft-embossing protocol may emerge as promising approach for manufacturing PMMA devices.
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Desenho de Equipamento/métodos , Dispositivos Lab-On-A-Chip , Procedimentos Analíticos em Microchip/métodos , Polimetil Metacrilato/química , Glicemia/análise , Colorimetria/instrumentação , Eletroforese/instrumentação , Temperatura Alta , Limite de Detecção , Modelos Lineares , Modelos Biológicos , Reprodutibilidade dos TestesRESUMO
This chapter describes the development of paper-based microchip electrophoresis (pME) devices for the separation of clinically relevant compounds. pME were fabricated by laser cut and thermal lamination process using polyester pouches. In addition, hand-drawn pencil electrodes were integrated to the device to perform capacitively coupled contactless conductivity detection (C4D). Finished device costs less than US$ 0.10 and did not require either sophisticated instrumentation or clean room facilities. Furthermore, pME is lightweight, easy to handle, flexible, and robust. pME-C4D device revealed an excellent capacity to separate BSA and creatinine in less than 150 s with baseline resolution. The device proposed in this chapter has proven to be a good alternative as a platform for the diagnosis of diseases from renal disorders such as diabetes mellitus and heart disease.
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Creatinina/análise , Eletroforese em Microchip/instrumentação , Desenho de Equipamento/métodos , Soroalbumina Bovina/análise , Animais , Bovinos , Diabetes Mellitus/diagnóstico , Condutividade Elétrica , Eletroforese em Microchip/métodos , Cardiopatias/diagnóstico , Humanos , Nefropatias/diagnóstico , Lasers , PapelRESUMO
This current review article focuses on recent contributions to on-site forensic investigations. Portable and potentially portable methods are presented and critically discussed about (bio)chemical trace analysis and studies performed outside the controlled laboratory environment to rapidly help in crime scene inquiries or forensic intelligence purposes. A wide range of approaches including electrochemical sensors, microchip electrophoresis, ambient ionization on portable mass spectrometers, handheld Raman and NIR instruments as well as and point-of-need devices, like paper-based platforms, for in-field analysis of latent evidences, controlled substances, drug screening, hazards, and others to assist in law enforcements and solving crime more efficiently are highlighted. The covered examples have successfully demonstrated the huge potential of portable devices for on-site applications. Future investigations should consider analytical validation to compete equality and even replace current gold standard methods.
Assuntos
Técnicas Eletroquímicas , Eletroforese em Microchip , Ciências Forenses/instrumentação , Drogas Ilícitas/análise , PapelRESUMO
This study describes the use of electrochemical paper-based analytical devices (ePADs) drawn with graphite pencil for the determination of ascorbic acid (AA) in commercial tablets. ePADs were fabricated using vegetal paper and graphite pencil. First, the three-electrode electrochemical cell drawn using a graphical software and toner lines were laser printed on the vegetal paper surface to delimit the electrode areas. Then, the electrode regions were manually painted with graphite pencil. Afterwards, the pseudo-reference electrode was defined with the deposition of silver ink over the graphite surface. Cyclic voltammetry and square wave voltammetry (SWV) experiments were performed to optimize the electroanalytical parameters as well as to quantitatively determine the AA concentration in two commercial tables. ePADs exhibited linear behavior for a concentration range between 0.5 and 3.0 mmol L-1. The achieved limit of detection and sensitivity were 70 µmol L-1 and 0.47 µA/mmol L-1, respectively. The AA concentration levels found by SWV experiments in both CenevitTM and Energil CTM were 2.80 ± 0.02 and 3.10 ± 0.01 mmol L-1, respectively. The accuracy of the proposed devices was investigated through recovery experiments in three concentration levels and it presented values between 95 and 115%.
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Ácido Ascórbico/análise , Eletroquímica/instrumentação , Papel , Ácido Ascórbico/química , Eletrodos , Grafite/químicaRESUMO
A colorimetric point-of-care paper-based analytical device (PAD) is developed for detecting adulterated beverages using whiskey falsified with caramel color as a model. Combining principal component analysis and calibration curves facilitated identification of adulteration in samples seized by the Brazilian Federal Police, at only â¼$0.02 per sample.
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Simple methods have been developed for fabricating microfluidic paper-based analytical devices (µPADs) but few of these devices can be used with organic solvents and/or aqueous solutions containing surfactants. This study describes a simple fabrication strategy for µPADs that uses readily available scholar glue to create the hydrophobic flow barriers that are resistant to surfactants and organic solvents. Microfluidic structures were defined by magnetic masks designed with either neodymium magnets or magnetic sheets to define the patter, and structures were created by spraying an aqueous solution of glue on the paper surface. The glue-coated paper was then exposed to UV/Vis light for cross-linking to maximize chemical resistance. Examples of microzone arrays and microfluidic devices are demonstrated. µPADs fabricated with scholar glue retained their barriers when used with surfactants, organic solvents, and strong/weak acids and bases unlike common wax-printed barriers. Paper microzones and microfluidic devices were successfully used for colorimetric assays of clinically relevant analytes commonly detected in urinalysis to demonstrate the low background of the barrier material and generally applicability to sensing. The proposed fabrication method is attractive for both its ability to be used with diverse chemistries and the low cost and simplicity of the materials and process.
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This chapter describes two different methodologies used to improve the analytical performance of colorimetric paper-based biosensors. Microfluidic paper-based analytical devices (µPADs) have been produced by a stamping process and CO2 laser ablation and modified, respectively, through an oxidation step and incorporation of silica nanoparticles on the paper structure. Both methods are employed in order to overcome the largest problem associated with colorimetric detection, the heterogeneity of the color distribution in the detection zones. The modification steps are necessary to improve the interaction between the paper surface and the selected enzymes. The enhanced performance has ensured reliability for quantitative analysis of clinically relevant compounds.
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Técnicas Biossensoriais/métodos , Colorimetria/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Nanopartículas , Papel , Bioensaio/instrumentação , Bioensaio/métodos , Biomarcadores/urina , Técnicas Biossensoriais/instrumentação , Colorimetria/instrumentação , Desenho de Equipamento , Humanos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , OxirreduçãoRESUMO
This paper describes the modification of microfluidic paper-based analytical devices (µPADs) with chitosan to improve the analytical performance of colorimetric measurements associated with enzymatic bioassays. Chitosan is a natural biopolymer extensively used to modify biosensing surfaces due to its capability of providing a suitable microenvironment for the direct electron transfer between an enzyme and a reactive surface. This hypothesis was investigated using glucose and uric acid (UA) colorimetric assays as model systems. The best colorimetric sensitivity for glucose and UA was achieved using a chromogenic solution composed of 4-aminoantipyrine and sodium 3,5-dichloro-2-hydroxy-benzenesulfonate (4-AAP/DHBS), which provided a linear response for a concentration range between 0.1 and 1.0 mM. Glucose and UA were successfully determined in artificial serum samples with accuracies between 87 and 114%. The limits of detection (LODs) found for glucose and UA assays were 23 and 37 µM, respectively. The enhanced analytical performance of chitosan-modified µPADs allowed the colorimetric detection of glucose in tear samples from four nondiabetic patients. The achieved concentration levels ranged from 130 to 380 µM. The modified µPADs offered analytical reliability and accuracy as well as no statistical difference from the values achieved through a reference method. Based on the presented results, the proposed µPAD can be a powerful alternative tool for non-invasive glucose analysis.
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Quitosana , Colorimetria , Glucose/análise , Dispositivos Lab-On-A-Chip , Papel , Ácido Úrico/análise , Humanos , Técnicas Analíticas Microfluídicas , Reprodutibilidade dos Testes , Lágrimas/químicaRESUMO
Th1 immune responses are crucial for eliminating Leishmania parasites. However, despite strong Th1 responses, cutaneous leishmaniasis (CL) patients infected with Leishmania braziliensis develop the disease, while milder Th1 responses are found in sub-clinical (SC) infections. Therefore, CL patients may experience impaired regulatory T cell (Treg) function, causing excessive Th1 responses and tissue damage. To address this hypothesis, we characterized the function of circulating Tregs in L. braziliensis infected CL patients and compared them to Tregs from uninfected controls (UC) and SC subjects. The frequency of circulating Tregs was similar in CL patients, UC and SC subjects. Moreover, CL patients Tregs suppressed lymphocyte proliferation and PBMC pro-inflammatory cytokine production more efficiently than UC Tregs, and also produced higher levels of IL-10 than UC and SC Tregs. Furthermore, PBMC and mononuclear cells from lesions of CL patients responded normally to Treg-induced suppression. Therefore, the lesion development in CL patients infected with L. braziliensis is not associated with impairment in Treg function or failure of cells to respond to immunomodulation. Rather, the increased Treg activation in CL patients may impair parasite elimination, resulting in establishment of chronic infection. Thus, immunological strategies that interfere with this response may improve leishmaniasis treatment.
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Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Idoso , Antígenos CD4/metabolismo , Estudos de Casos e Controles , Criança , Citocinas/metabolismo , Feminino , Humanos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/metabolismo , Adulto JovemRESUMO
BACKGROUND: Obesity has been associated with the severity of psoriasis, but this relationship is not completely understood. OBJECTIVES: This study aimed to evaluate associations between the severity of psoriasis and weight excess as determined using a variety of parameters. METHODS: A cross-sectional study was performed in 296 psoriasis patients. Their body mass index (BMI), waist circumference (WC), and waist : hip ratio (WHR) values were compared with results on a psoriasis area severity index (PASI). RESULTS: The frequency of severe psoriasis was higher in men (P < 0.05). Direct correlations were established between PASI scores and each of BMI (R = 0.0154, P = 0.01), WC (R = 0.207, P = 0.001), and WHR (R = 0.164, P = 0.007). CONCLUSIONS: This study extends previous reports of an association between psoriasis and obesity and shows a direct correlation between obesity as measured according to different parameters and psoriasis severity.
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Índice de Massa Corporal , Obesidade/epidemiologia , Psoríase/epidemiologia , Índice de Gravidade de Doença , Adulto , Comorbidade , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sobrepeso/epidemiologia , Prevalência , Distribuição por Sexo , Circunferência da Cintura , Relação Cintura-QuadrilRESUMO
Human T cell lymphotropic virus type-1 (HTLV-1) induces activation and spontaneous proliferation of T cells with production of type-1 pro-inflammatory cytokines. It modifies the immune response to other antigens and increases susceptibility to infectious diseases. However, little is known about innate immunity in HTLV-1 infection. HTLV-1-infected individuals have higher spontaneous neutrophil activation than HTLV-1-seronegative individuals, as shown by the nitroblue tetrazolium (NBT) assay. This study was conducted to evaluate neutrophil function in HTLV-1-infected individuals. Participants in the study included 18 HTLV-1-infected individuals and 14 HTLV-1-seronegative controls. We evaluated the ability of neutrophils (PMNs) to control a parasite infection, to produce peroxynitrite, cytokines and chemokines and to express activation markers in cultures when stimulated with LPS or infected with Leishmania. When compared with the control group, there was no difference in the percentage of PMNs infected with Leishmania or in the number of amastigotes/100 PMNs in HTLV-1-infected individuals. The microbicidal activity of the PMNs and the levels of CXCL8 and CCL4 released by these cells did not show a difference between HTLV-1-infected individuals and the control group. In both the HTLV-1 group and the control group, infection with Leishmania or stimulation of PMNs led to cellular activation. These observations suggest that neutrophils from HTLV-1-infected individuals have preserved their ability to become activated and to produce chemokines and peroxynitrite after stimulation and that the susceptibility to infection by intracellular Leishmania amazonensis in HTLV-1-infected individuals does not depend on impairment of neutrophil function.
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Quimiocinas/imunologia , Citocinas/imunologia , Infecções por HTLV-I/imunologia , Leishmania mexicana/imunologia , Neutrófilos/imunologia , Neutrófilos/parasitologia , Antígenos CD/biossíntese , Moléculas de Adesão Celular/biossíntese , Quimiocinas/biossíntese , Citocinas/biossíntese , Feminino , Proteínas Ligadas por GPI/biossíntese , Humanos , Imunidade Inata , Selectina L/biossíntese , Lipopolissacarídeos/imunologia , Masculino , Ativação de Neutrófilo , Neutrófilos/metabolismo , Ácido Peroxinitroso/biossíntese , Explosão RespiratóriaRESUMO
O objetivo deste trabalho foi comparar, in vitro, diferentes técnicas de acabamento de preparos por meio da análise da rugosidade superficial e microscopia eletrônica de varredora (MEV). Cinquenta e seis pré-molares humanos foram divididos em quatro grupos, (n= 10): Grupo I - sem acabamento (grupo controle); Grupo II - ponta diamantada 2135 F e 2135 FF; Grupo III - ponta 2135 acopladas em um contra ângulo multiplicador (Kavo Koncept 1:5) e Grupo IV - ponta CVD cilíndrica 8.2137 (CVDentus- Clorovale) em aparelho de ultra-som (Profi II - Dabi Atlante). Após o acabamento, dez espécimes de cada grupo foram submetidos à leitura rugosimétrica e quatro foram analisados no MEV (Microscopia eletrônica de varredura). Sessenta e quatro micrografias foram analisadas por doze examinadores. Os valores de Ra (média±dp) foram: GI- 1,46±0,2; GII- 0,87±0,3; GIII- 1,6±0,4 e GIV- 1,34±0,2. Após teste de t-Student, observou-se que não houve diferença significativa entre os grupos I (controle), III (multiplicador) e IV (CVD). Entretanto, houve diferença entre o grupo I e II; Grupo II e III; GII e I. Após teste de Mann-Whitney para análise em MEV, observou-se que não houve diferença significativa entre os grupos I (controle), II (ponta diamantada fina e extra-fina) e III (multiplicador). Conclui-se que o grupo que apresentou menor valor de rugosidade (Ra) foi o grupo II, realizado com pontas diamantadas 2135 F e 2135FF montadas em alta rotação. Para análise em MEV, concluiu-se que a superfície produzida pelo grupo IV (CVD) foi a mais rugosa.
The objective of this study was to compare, in vitro, different techniques for finishing preparations of facets indirect through the analysis of surface roughness and electron microscopy sweeper (SEM). Forty human premolars were divided into 4 groups according to the finishing of the cavity preparation for indirect facets: Group I (n = 10) - unfinished (control group), Group II (n = 10) - diamond bur 2135 F and 2135FF, Group III (n = 10)- tip in 2135 engaged in a multiplier (Contra angle multiplier Koncept Kavo 1:5) and Group IV (n = 10) tip cylindrical CVDcat. 8.2137 (CVDentus - Clorovale) in equipment ultrasound (Profi II- Dabi Atlante). The reading was held rugosimetric relief meter Mitutoyo SJ-201. For SEM analysis were prepared 16 teeth were divided into four groups according to the finishing technique described above, with four teeth in each group, totaling 64 images, analyzed by twelve examiners. The values of Ra (mean ± SD) were: GI-1.46 ± 0.2, GII - 0.87 ± 0.3, GIII, 1.6 ± 0.4 and GIV-1.34 ± 0.2 . After test t-test (Figure 1), it was observed that there was no significant difference between groups I (control),III (multiplier) and IV (CVD). However, there was difference between group I and II, Group II and III, GII and I. After the Mann-Whitney test for analysis by SEM, it was observed that there was no significant difference between groups I (control), II (thin diamond point) and III (multiplier). However, there was difference between group I, II, III with group IV (CVD), with p <0.001. It follows that the group with lowest roughness (Ra) (smoother) the group II was performed with diamond burs and 2135 F 2135FF mounted on heavy rotation. For SEM analysis, itwas concluded that surface produced by the group IV (CVD) was the most wrinkled. The values assigned by examiners through the analysis of SEM were low averaging less than 5.0 for all groups.
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Interleukin 17 (IL-17) plays a critical role in inflammation and autoimmunity. Very little is known about IL-17 in protozoa infection. Here, we show that lymphocytes obtained from patients with mucosal leishmaniasis and cutaneous leishmaniasis produce higher levels of IL-17 than do lymphocytes obtained from uninfected control subjects (P<.01). There was a tendency for tissue obtained from patients with mucosal leishmaniasis to contain a higher number of cells expressing IL-17, compared with tissue obtained from patients with cutaneous leishmaniasis, and there was a direct correlation between the number of cells expressing IL-17 and the presence of cellular inflammation at the lesion site (r2 = 0.86; P<.001) These data support the role of IL-17 in the pathogenesis of the inflammatory reaction in leishmaniasis.