Perspectives for the use of latex peptidases from Calotropis procera for control of inflammation derived from Salmonella infections.

BACKGROUND: Anti-inflammatory properties have been attributed to latex proteins of the medicinal plant Calotropis procera. PURPOSE: A mixture of cysteine peptidases (LPp2) from C. procera latex was investigated for control of inflammatory mediators and inflammation in a mouse model of Salmonella infection. METHODS: LPp2 peptidase activity was confirmed by the BANA assay. Cytotoxicity assays were conducted with immortalized macrophages. Peritoneal macrophages (pMØ) from Swiss mice were stimulated with lipopolysaccharide (LPS) in 96-well plates and then cultured with nontoxic concentrations of LPp2. Swiss mice intravenously received LPp2 (10 mg/kg) and then were challenged intraperitoneally with virulent Salmonella enterica Ser. Typhimurium. RESULTS: LPp2 was not toxic at dosages lower than 62.2 µg/mL. LPp2 treatments of pMØ stimulated with LPS impaired mRNA expression of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6 and IL-10. LPp2 increased the intracellular bacterial killing in infected pMØ. Mice given LPp2 had a lower number of leukocytes in the peritoneal cavity in comparison to control groups 6 h after infection. The bacterial burden and histological damage were widespread in target organs of mice receiving LPp2. CONCLUSION: We conclude that LPp2 contains peptidases with strong anti-inflammatory properties, which may render mice more susceptible to early disseminated infection caused by Salmonella.

Latex protein extracts from Calotropis procera with immunomodulatory properties protect against experimental infections with Listeria monocytogenes.

BACKGROUND: The latex from the medicinal plant Calotropis procera is often used in folk medicine against infectious and inflammatory diseases. PURPOSE: In this study, we investigate a protein fraction with immunomodulatory properties, named LPPI, against experimental infections, in vitro and in vivo, with a virulent strain of Listeria monocytogenes. STUDY DESIGN: LPPI was exposed to cultured macrophages or Swiss mice and then challenged with L. monocytogenes. METHODS: Peritoneal macrophages were obtained from Swiss mice, and cultured in 96-well microplates. Soluble latex proteins (LP) were subjected to fractionation by ion-exchange chromatography. The major peak (LPPI) was added into wells at 10 or 100µg/ml. Albumin (100µg/ml) was used for comparison between protein treatments. After incubation for 1h at 5% CO2/ 37°C, the supernatant was discarded and 0.2ml of L. monocytogenes overnight culture was added in the wells. Following 4h and 24h infection, the cytokine mRNA expression was evaluated as well as the number of intracellular colony forming units. Swiss mice (n=16) were injected intraperitoneally (i.p.) with LPPI (5 and 10mg/kg) while the control mice received albumin (10mg/kg) or LP (10mg/kg). After 24h, all animal groups were challenged with L. monocytogenes (10(6) CFU/ ml), also by i.p. route. RESULTS: LPPI was not toxic to uninfected macrophages (pMØ) and significantly increased mRNA expression of TNF-α, IL-6, IL-1ß and iNOS. Following infection, cell viability was reduced by 50% in albumin-treated pMØ (control); but only 17% in pMØ treated with LPPI at 100µg/ml. In this case, LPPI increased expression of TNF-α and IL-6 whereas the number of bacterial colony-forming units was reduced 100-fold in comparison to control groups. Swiss mice pretreated with LPPI showed dose-dependent survival rates that reached 80%, while mice that received albumin died 1-3 days after infection. After 24h infection, leukocyte migration to the infectious foci was high in LPPI-treated mice whereas the number of viable bacteria in the peritoneal fluid, liver and bloodstream were significantly reduced. CONCLUSION: We conclude that LPPI present immunomodulatory properties that are beneficial for prevention of systemic bacterial infections caused by the intracellular bacteria L. monocytogenes.

Peptidases from latex of Carica candamarcensis upregulate COX-2 and IL-1 mRNA transcripts against Salmonella enterica ser. Typhimurium-mediated inflammation.

The immunomodulatory properties of a mixture of cysteine peptidases (P1G10) obtained from the fruit lattice of Carica candamarcensis were investigated. P1G10 was obtained from fresh latex samples by chromatography in a Sephadex column and initially administered to Swiss mice (n = 5; 1 or 10 mg/kg) via i.p. After 30 min, the mice were injected with carrageenan (0.5 mg/mouse) or heat-killed S. Typhimurium (10(7) CFU/mL; 100°C/30 min) into the peritoneal cavity. Afterwards, two animal groups were i.p. administered with P1G10 (n = 6; 1, 5, or 10 mg/Kg) or PBS 24 hours prior to challenge with live S. Typhimurium (10(7) CFU/mL). P1G10 stimulated the proliferation of circulating neutrophils and lymphocytes, 6 h after injection of carrageenan or heat-killed bacteria, respectively. Furthermore, survival after infection was dose-dependent and reached 60% of the animal group. On the other hand, control mice died 1-3 days after infection. The examination of mRNA transcripts in liver cells 24 h after infection confirmed fold variation increases of 5.8 and 4.8 times on average for IL-1 and COX-2, respectively, in P1G10 pretreated mice but not for TNF-α, IL-10, γ-IFN and iNOS, for which the results were comparable to untreated animals. These data are discussed in light of previous reports.

Phytochemistry and Preliminary Assessment of the Antibacterial Activity of Chloroform Extract of Amburana cearensis (Allemão) A.C. Sm. against Klebsiella pneumoniae Carbapenemase-Producing Strains.

The chloroform extract of the stem bark of Amburana cearensis was chemically characterized and tested for antibacterial activity.The extract was analyzed by gas chromatography and mass spectrometry. The main compounds identified were 4-methoxy-3-methylphenol (76.7%), triciclene (3.9%), α -pinene (1.0%), ß -pinene (2.2%), and 4-hydroxybenzoic acid (3.1%). Preliminary antibacterial tests were carried out against species of distinct morphophysiological characteristics: Escherichia coli, Salmonella enterica Serotype Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. The minimum inhibitory concentration (MIC) was determinate in 96-well microplates for the chloroform extract and an analogue of themain compound identified, which was purchased commercially.We have shown that plant's extract was only inhibitory (but not bactericidal) at the maximum concentration of 6900 µ g/mL against Pseudomonas aeruginosa and Bacillus cereus. Conversely, the analogue 2-methoxy-4-methylphenol produced MICs ranging from215 to 431 µ g/mL against all bacterial species.New antibacterial assays conducted with such chemical compound against Klebsiella pneumoniae carbapenemase-producing strains have shown similarMICresults and minimumbactericidal concentration (MBC) of 431 µ g/mL.We conclude that A. cearensis is a good source of methoxy-methylphenol compounds,which could be screened for antibacterial activity againstmultiresistant bacteria fromdifferent species.

Zoonotic potential of multidrug-resistant extraintestinal pathogenic Escherichia coli obtained from healthy poultry carcasses in Salvador, Brazil

The zoonotic potential to cause human and/or animal infections among multidrug-resistant extraintestinal pathogenic Escherichia coli from avian origin was investigated. Twenty-seven extraintestinal pathogenic E. coli isolates containing the increased survival gene (iss) were obtained from the livers of healthy and diseased poultry carcasses at two slaughterhouses in Salvador, northeastern Brazil. The antimicrobial resistance-susceptibility profiles were conducted with antibiotics of avian and/or human use by the standardized disc-diffusion method. Antimicrobial resistance was higher for levofloxacin (51.8%), amoxicillin/clavulanic acid (70.4%), ampicillin (81.5%), cefalotin (88.8%), tetracycline (100%) and streptomycin (100%). The minimum inhibitory concentrations above the resistance breakpoints of doxycycline, neomycin, oxytetracycline and enrofloxacin reached, respectively, 88.0%, 100%, 75% and 91.7% of the isolates. Strains with high and low antimicrobial resistance were i.p. administered to Swiss mice, and histopathological examination was carried out seven days after infection. Resistance to goat and human serum complement was also evaluated. The results show that Swiss mice challenged with strain 2B (resistant to 11 antimicrobials) provoked a severe degeneration of hepatocytes besides lymphocytic infiltration in the liver, whereas the spleen showed areas of degeneration of the white and red pulp. Conversely, the spleen and liver of mice challenged with strain 4A (resistant to two antimicrobials) were morphologically preserved. In addition, complement resistance to goat and human serum was high for strain 2B and low for strain 4A. Our data show that multidrug resistance and pathogenesis can be correlated in extraintestinal pathogenic E. coli strains obtained from apparently healthy poultry carcasses, increasing the risk for human public healthy.

Zoonotic potential of multidrug-resistant extraintestinal pathogenic Escherichia coli obtained from healthy poultry carcasses in Salvador, Brazil.

The zoonotic potential to cause human and/or animal infections among multidrug-resistant extraintestinal pathogenic Escherichia coli from avian origin was investigated. Twenty-seven extraintestinal pathogenic E. coli isolates containing the increased survival gene (iss) were obtained from the livers of healthy and diseased poultry carcasses at two slaughterhouses in Salvador, northeastern Brazil. The antimicrobial resistance-susceptibility profiles were conducted with antibiotics of avian and/or human use by the standardized disc-diffusion method. Antimicrobial resistance was higher for levofloxacin (51.8%), amoxicillin/clavulanic acid (70.4%), ampicillin (81.5%), cefalotin (88.8%), tetracycline (100%) and streptomycin (100%). The minimum inhibitory concentrations above the resistance breakpoints of doxycycline, neomycin, oxytetracycline and enrofloxacin reached, respectively, 88.0%, 100%, 75% and 91.7% of the isolates. Strains with high and low antimicrobial resistance were i.p. administered to Swiss mice, and histopathological examination was carried out seven days after infection. Resistance to goat and human serum complement was also evaluated. The results show that Swiss mice challenged with strain 2B (resistant to 11 antimicrobials) provoked a severe degeneration of hepatocytes besides lymphocytic infiltration in the liver, whereas the spleen showed areas of degeneration of the white and red pulp. Conversely, the spleen and liver of mice challenged with strain 4A (resistant to two antimicrobials) were morphologically preserved. In addition, complement resistance to goat and human serum was high for strain 2B and low for strain 4A. Our data show that multidrug resistance and pathogenesis can be correlated in extraintestinal pathogenic E. coli strains obtained from apparently healthy poultry carcasses, increasing the risk for human public healthy.