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Registros recuperados: 31
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A method for multiple sequential analyses of macrophage functions using a small single cell sample BJMBR
Nascimento,F.R.F.; Rodríguez,D.; Gomes,E.; Fernvik,E.C.; Russo,M..
Microbial pathogens such as bacillus Calmette-Guérin (BCG) induce the activation of macrophages. Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II). Multiple microassays have been developed to measure these parameters. Usually each assay requires 2-5 x 10(5) cells per well. In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages. Here we describe a method whereby this limitation can be circumvented. Using a single 96-well microassay and a very small...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Macrophages; Nitric oxide; Hydrogen peroxide; MHC II; Method.
Ano: 2003 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2003000900012
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Classical and alternative macrophages have impaired function during acute and chronic HIV-1 infection BJID
Galvão-Lima,Leonardo J.; Espíndola,Milena S.; Soares,Luana S.; Zambuzi,Fabiana A.; Cacemiro,Maira; Fontanari,Caroline; Bollela,Valdes R.; Frantz,Fabiani G..
Abstract Objectives: Three decades after HIV recognition and its association with AIDS development, many advances have emerged – especially related to prevention and treatment. Undoubtedly, the development of Highly Active Antiretroviral Therapy (HAART) dramatically changed the future of the syndrome that we know today. In the present study, we evaluate the impact of Highly Active Antiretroviral Therapy on macrophage function and its relevance to HIV pathogenesis. Methods: PBMCs were isolated from blood samples and monocytes (CD14+ cells) were purified. Monocyte-Derived Macrophages (MDMs) were activated on classical (MGM-CSF+IFN-γ) or alternative (MIL-4+IL13) patterns using human recombinant cytokines for six days. After this period, Monocyte-Derived...
Tipo: Info:eu-repo/semantics/article Palavras-chave: HIV-1; Innate immune response; HAART; Macrophages.
Ano: 2017 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702017000100042
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Cytotoxic activity of BCG-activated macrophages against L929 tumor cells is nitric oxide-dependent BJMBR
Nascimento,F.R.F.; Ribeiro-Dias,F.; Russo,M..
The tumoricidal activity of activated macrophages has been attributed largely to the release of tumor necrosis factor (TNF), or to the production of reactive oxygen or nitrogen intermediates. The L929 tumor cell line (a murine fibroblast-like cell) when treated with actinomycin D (ActD) has been used to measure TNF<FONT FACE="Symbol">a</FONT> cytotoxicity. In the present study, we determined the cytotoxic activity of BCG-activated peritoneal macrophages against ActD-untreated L929 tumor cells. Furthermore, we measured the production of hydrogen peroxide (H2O2), nitric oxide (NO) and TNF by macrophages cultured in the presence or absence of L929 cells. As expected, BCG-activated macrophages produced significant amounts of H2O2 (16.0 ± 3.0 µM),...
Tipo: Info:eu-repo/semantics/other Palavras-chave: Cytotoxicity; Macrophages; L929 tumor cells; Nitric oxide; TNF; Hydrogen peroxide.
Ano: 1998 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1998001200012
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Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction BJMBR
Bonacorsi,C.; Raddi,M.S.G.; Carlos,I.Z..
Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Chlorhexidine; Cytotoxicity; Macrophages; Nitric oxide; Hydrogen peroxide.
Ano: 2004 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000200007
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Depletion of Phagocytic Cells during Nonlethal Plasmodium yoelii Infection Causes Severe Malaria Characterized by Acute Renal Failure in Mice OAK
Terkawi, Mohamad Alaa; Nishimura, Maki; Furuoka, Hidefumi.
In the current study, we examined the effects of depletion of phagocytes on the progression of Plasmodium yoelii 17XNL infection in mice. Strikingly, the depletion of phagocytic cells, including macrophages, with clodronate in the acute phase of infection significantly reduced peripheral parasitemia but increased mortality. Moribund mice displayed severe pathological damage, including coagulative necrosis in liver and thrombi in the glomeruli, fibrin deposition, and tubular necrosis in kidney. The severity of infection was coincident with the increased sequestration of parasitized erythrocytes, the systematic upregulation of inflammation and coagulation, and the disruption of endothelial integrity in the liver and kidney. Aspirin was administered to the...
Palavras-chave: Phagocytes; Macrophages; Phagocytosis; Plasmodium yoelii.
Ano: 2016 URL: http://ir.obihiro.ac.jp/dspace/handle/10322/4479
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Determination of the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro: MGIT 960 as a viable alternative for BACTEC 460 BJID
Jhamb,Sarbjit Singh; Goyal,Amit; Singh,Prati Pal.
BACTEC 460 has now been phased out, so the search for an alternative is imperative. We have determined the activity of standard anti-tuberculosis drugs against intramacrophage Mycobacterium tuberculosis, in vitro, by using BACTEC 460 and MGIT 960 methods. The minimum inhibitory concentrations of isoniazid, rifampicin, ethambutol and streptomycin against intracellular M. tuberculosis H37Rv were found to be 0.2, 0.8, 8.0, and 5.0 µg/mL, respectively, by both methods. These results show a significant (p < 0.001) concordance between minimum inhibitory concentrations obtained by these two different methods. MGIT 960 system uses a robust florescence quenching-based oxygen sensor, requires no radioisotope, is safe, and relatively easy to operate. Apparently,...
Tipo: Info:eu-repo/semantics/other Palavras-chave: BACTEC 460; Macrophages; MGIT 960; Mycobacterium tuberculosis.
Ano: 2014 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702014000300336
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Differential roles for pathogenicity islands SPI-13 and SPI-8 in the interaction of Salmonella Enteritidis and Salmonella Typhi with murine and human macrophages Biol. Res.
Espinoza,Rodrigo A.; Silva&#8209;Valenzuela,Cecilia A.; Amaya,Fernando A.; Urrutia,Ítalo M.; Contreras,Inés; Santiviago,Carlos A..
Abstract Background Salmonella pathogenicity island (SPI)-13 is conserved in many serovars of S. enterica, including S. Enteritidis, S. Typhimurium and S. Gallinarum. However, it is absent in typhoid serovars such as S. Typhi and Paratyphi A, which carry SPI-8 at the same genomic location. Because the interaction with macrophages is a critical step in Salmonella pathogenicity, in this study we investigated the role played by SPI-13 and SPI-8 in the interaction of S. Enteritidis and S. Typhi with cultured murine (RAW264.7) and human (THP-1) macrophages. Results Our results showed that SPI-13 was required for internalization of S. Enteritidis in murine but not human macrophages. On the other hand, SPI-8 was not required for the interaction of S. Typhi...
Tipo: Journal article Palavras-chave: Salmonella; Enteritidis; Typhi; SPI-13; SPI-8; Macrophages; RAW264.7; THP-1.
Ano: 2017 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602017000100402
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Effect of Bothrops alternatus snake venom on macrophage phagocytosis and superoxide production: participation of protein kinase C J. Venom. Anim. Toxins incl. Trop. Dis.
Setubal,SS; Pontes,AS; Furtado,JL; Kayano,AM; Stábeli,RG; Zuliani,JP.
Envenomations caused by different species of Bothrops snakes result in severe local tissue damage, hemorrhage, pain, myonecrosis, and inflammation with a significant leukocyte accumulation at the bite site. However, the activation state of leukocytes is still unclear. According to clinical cases and experimental work, the local effects observed in envenenomation by Bothrops alternatus are mainly the appearance of edema, hemorrhage, and necrosis. In this study we investigated the ability of Bothrops alternatus crude venom to induce macrophage activation. At 6 to 100 ¼g/mL, BaV is not toxic to thioglycollate-elicited macrophages; at 3 and 6 ¼g/mL, it did not interfere in macrophage adhesion or detachment. Moreover, at concentrations of 1.5, 3, and 6 ¼g/mL...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Snake venom; Bothrops; Macrophages; Phagocytosis; Superoxide; Protein kinase C; Inflammation.
Ano: 2011 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992011000400010
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Effect of citrinin and in association with aflatoxin B1 on the infectivity and proliferation of Toxoplasma gondii in vitro BJID
Herzog-Soares,Joanna D'Arc A; Freire,Ronald Bastos.
Macrophages exposed to 10 mug/mL citrinin (CTR) or 0.01 mug CTR mixed with 0.04 mug aflatoxin B1 (AFB1) for a period of 2 h at 37ºC, were infected with 10(6) Toxoplasma gondii tachyzoites/muL. The parasites were treated with mycotoxins (2 h at 37ºC) before being added to the macrophage culture. The number of tachyzoites was quantified 2, 24, 48, 72 and 96 h after infection. During the first 2 hours, 59% infectivity was observed in the control. After exposure to CTR or the mixture of toxins (CTR-AFB1), macrophages were infected with 77.5% and 75% of the inoculated tachyzoites, respectively. Similarly, 72.3% of the cells were infected when cultured together with previously treated parasites. The treatment with CTR-AFB1 gave rise to 2.9 times more tachyzoites...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Aflatoxins; Imunossuspression; Macrophages; Citrinin; Toxoplasma gondii.
Ano: 2004 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702004000100007
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Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis J. Venom. Anim. Toxins incl. Trop. Dis.
Bordon,Maria L. A. C.; Laurenti,Márcia D.; Ribeiro,Susan Pereira; Toyama,Marcos H.; Toyama,Daniela de O.; Passero,Luiz Felipe D..
Abstract Background: Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.)...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Leishmania (Leishmania) amazonensis; Macrophages; BALB/c mice; Phospholipase A2; Phospholipase A2inhibitors.
Ano: 2018 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992018000100312
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Effects of diazepam on Mycobacterium bovis-induced infection in hamsters BJMBR
Righi,D.A.; Pinheiro,S.R.; Guerra,J.L.; Palermo-Neto,J..
The in utero exposure of hamsters to low doses of diazepam results in impaired host defense against Mycobacterium bovis during adulthood. Delayed developmental immunotoxicity, however, represents a specific situation that might not be general. The present experiment was undertaken to investigate the effects of diazepam on hamster resistance to M. bovis using adult animals. The effects of diazepam treatment on serum cortisol levels were also studied. Adult hamsters (N = 10 for each group) were treated with diazepam (E1 = 1.0, E2 = 2.0 or E3 = 3.0 mg kg-1 day-1 subcutaneously) or with control solution (C) for 30 days. Seven days after the beginning of the treatment, the animals received identical inoculum concentrations of M. bovis. Hamsters treated with the...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Mycobacterium bovis; Host resistance; Macrophages; Diazepam; Immune system.
Ano: 1999 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1999000900014
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Gene expression in IFN-gamma-activated murine macrophages BJMBR
Pereira,C.A.; Modolell,M.; Frey,J.R.; Lefkovits,I..
Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Gene array; MRNA; Mouse hepatitis virus 3 (MHV3); IFN-gamma; BALB/c mouse; A/J mouse; Macrophages.
Ano: 2004 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001200005
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Immunohistochemical Studies on Duodenum, Spleen and Liver in Mice: Distribution of Ferroportin and Prohepcidin in an Inflammation Model International Journal of Morphology
D´Anna,María Cecilia; Giorgi,Gisela; Roque,Marta Elena.
Duodenum, spleen and liver have a crucial role in iron balance on the whole organism and are the major sites of Ferroportin (FPN) expression. Specific regulations between FPN and hepcidin are responsible for changes seen in physiopathological conditions such as inflammation. We studied in vivo effects of turpentine oil-induced acute inflammation on FPN expression, and its relation with prohepcidin and iron mobilization. Immunohistochemical procedures were performed using rabbit anti-mouse FPN and prohepcidin antibodies with goat-labeled polymer-HRP anti-rabbit (DAB) as secondary antibody. Plasma and tissular iron were also studied. Our results showed a notable expression and redistribution of duodenal FPN to basolateral membrane in turpentine-treated mice,...
Tipo: Journal article Palavras-chave: Iron; Mobilization; Macrophages; Enterocytes.
Ano: 2011 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-95022011000300014
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Immunomodulatory action of propolis on macrophage activation J. Venom. Anim. Toxins
ORSI,R.O.; FUNARI,S.R.C.; SOARES,A.M.V.C.; CALVI,S.A.; OLIVEIRA,S.L.; SFORCIN,J.M.; BANKOVA,V..
Propolis has been the subject of several recent studies, with the aim of elucidating its biological and pharmacological properties. Propolis has a well-known antimicrobial activity as well as antioxidant, antitumoral, antiinflammatory, and regenerative properties, but literature about its effects on the immune response is scarce. The goal of this work was to evaluate the propolis effect on macrophage activation by oxygen (H2O2) and nitrogen (NO) metabolite determination. Propolis was produced by africanized honeybees and hydroalcoholic solutions were prepared at different concentrations. Peritoneal macrophages were obtained from male BALB/c mice and culture cells were stimulated in vitro with propolis or interferon-gamma (IFN-gamma). In the in vivo assay,...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Propolis; Macrophages; Hydrogen peroxide; Nitric oxide.
Ano: 2000 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0104-79302000000200006
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Induction of cell-mediated immunity during early stages of infection with intracellular protozoa BJMBR
Gazzinelli,R.T.; Talvani,A.; Camargo,M.M.; Santiago,H.C.; Oliveira,M.A.P.; Vieira,L.Q.; Martins,G.A.; Aliberti,J.C.S.; Silva,J.S..
Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-<FONT FACE="Symbol">g</font>. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Leishmania; Toxoplasma gondii; Trypanosoma cruzi; Macrophages; Cytokines; Nitric oxide.
Ano: 1998 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1998000100012
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Influence of Covering Reused Broiler Litter with Plastic Canvas on Litter Characteristics and Bacteriology and the Subsequent Immunity and Microbiology of Broilers Rev. Bras. Ciênc. Avic.
Mesa,D; Lourenço,M; Souza,A; Bueno,A; Pereira,A; Sfeir,M; Santin,E.
ABSTRACT In broiler production, the litter is reused for consecutives flocks, and it is treated during down time between flocks to reduce its microbial load. Although covering the litter with a plastic canvas is a common litter treatment in the field, there is little scientific information available on its efficacy. The aim of this study was to evaluate the effects of covering broiler litter with a plastic canvas for eight days on litter microbiological, physical, and chemical parameters, and on the intestinal microbiota and immunity of broilers. In the first trial, reused litter from a previous flock was distributed into three treatments, with six replicates each: L1 (negative control, litter free from Salmonella Enteritidis (SE) and Eimeria maxima (EM)...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Ammonia; Cytokines; Lactobacillus; Macrophages; Salmonella.
Ano: 2016 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-635X2016000400563
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Inhibitory effects of Zanthoxylum rhoifolium Lam. (Rutaceae) against the infection and infectivity of macrophages by Leishmania amazonensis Anais da ABC (AABC)
MELO NETO,BERNARDO; LEITÃO,JOSEANA M.S.R.; OLIVEIRA,LUCIANO G.C.; SANTOS,SÉRGIO E.M.; CARNEIRO,SABRINA M.P.; RODRIGUES,KLINGER A.F.; CHAVES,MARIANA H.; ARCANJO,DANIEL D.R.; CARVALHO,FERNANDO A.A..
ABSTRACT Zanthoxylum rhoifolium Lam. (Rutaceae) has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH) and its n-hexane fraction (ZR-FHEX) on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated with ZR-EEtOH or ZR-FHEX at non-lethal concentrations for 24 hours, and then macrophages were submitted to infection by promastigotes. Moreover, their effects on activation of macrophages, as well as on the DNA content, size and number of promastigotes by flow...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Leishmania; Macrophages; Nitric oxide; Promastigote; Phagocytic activity; Zanthoxylum.
Ano: 2016 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652016000501851
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Lactobacillus delbrueckii UFV-H2b20 induces type 1 cytokine production by mouse cells in vitro and in vivo BJMBR
Neumann,E.; Ramos,M.G.; Santos,L.M.; Rodrigues,A.C.P.; Vieira,E.C.; Afonso,L.C.C.; Nicoli,J.R.; Vieira,L.Q..
Lactobacillus delbrueckii UFV-H2b20 has been shown to increase clearance of bacteria injected into the blood of germ-free mice. Moreover, it induces the production of type 1 cytokines by human peripheral mononuclear cells. The objective of the present study was to investigate the production of inflammatory cytokines [interleukin-12 (IL-12 p40), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)] triggered in vitro by live, heat-killed or lysozyme-treated L. delbrueckii UFV-H2b20 and in vivo by a live preparation. Germ-free, L. delbrueckii-monoassociated and lipopolysaccharide (LPS)-resistant C3H/HeJ mice were used as experimental models. UFV-H2b20 induced the production of IL-12 p40 and TNF-α by peritoneal cells and IFN-γ by spleen cells from...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Probiotics; Cytokines; Macrophages; IFN-γ; IL-12; Lactobacillus.
Ano: 2009 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2009000400008
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Metabolic fate of glutamine in lymphocytes, macrophages and neutrophils BJMBR
Curi,R.; Newsholme,P.; Pithon-Curi,T.C.; Pires-de-Melo,M.; Garcia,C.; Homem-de-Bittencourt Jr.,P.I.; Guimarães,A.R.P..
Eric Newsholme's laboratory was the first to show glutamine utilization by lymphocytes and macrophages. Recently, we have found that neutrophils also utilize glutamine. This amino acid has been shown to play a role in lymphocyte proliferation, cytokine production by lymphocytes and macrophages and phagocytosis and superoxide production by macrophages and neutrophils. Knowledge of the metabolic fate of glutamine in these cells is important for the understanding of the role and function of this amino acid in the maintenance of the proliferative, phagocytic and secretory capacities of these cells. Glutamine and glucose are poorly oxidized by these cells and might produce important precursors for DNA, RNA, protein and lipid synthesis. The high rate of...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Glutamine; Lymphocytes; Macrophages; Neutrophils; Metabolic fate.
Ano: 1999 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1999000100002
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Metabotropic glutamate receptor 5 may be involved in macrophage plasticity Biol. Res.
Shanshiashvili,Lali; Tsitsilashvili,Elene; Dabrundashvili,Nino; Kalandadze,Irine; Mikeladze,David.
Abstract Background Macrophages are a functionally heterogeneous cell population and depending on microenvironments they polarize in two main groups: M1 and M2. Glutamic acid and glutamate receptors may participate in the regulation of macrophage plasticity. To investigate the role of glutamatergic systems in macrophages physiology, we performed the transfection of mGluR5 cDNAs into RAW-264.7 cells. Results Comparative analysis of modified (RAW-mGluR5 macrophages) and non-modified macrophages (RAW-macrophages) has shown that the RAW-mGluR5 macrophages absorbed more glutamate than control cells and the amount of intracellular glutamate correlated with the expression of excitatory amino acid transporters -2 (EAAT-2). Besides, our results have shown that...
Tipo: Journal article Palavras-chave: Macrophages; Metabotropic glutamate receptors; Inflammation; HMGB1; IL-10.
Ano: 2017 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602017000100203
Registros recuperados: 31
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