Recombinant osmotin inclusion bodies from Calotropis procera produced in E. coli BL21(DE3) prevent acute inflammation in a mouse model of listeriosis.

BACKGROUND: The osmotin from the medicinal plant Calotropis procera (CpOsm) has characteristics similar to adiponectin, a human protein with immunoregulatory actions. PURPOSE: This study aimed to investigate whether recombinant osmotin inclusion bodies from C. procera (IB/rCpOsm) produced in E. coli BL21(DE3) can prevent infection-induced inflammation. A virulent strain of Listeria monocytogenes was used as an infection model. METHODS: Cells of E. coli BL21(DE3) carrying the plasmid pET303-CpOsm were used to express the recombinant osmotin, which accumulated at reasonable levels as inclusion bodies (IB/rCpOsm). IB/rCpOsm were purified from induced cells and SDS-polyacrylamide gel electrophoresis followed by mass spectrometry analyses confirmed the identity of the major protein band (23 kDa apparent molecular mass) as CpOsm. Peritoneal macrophages (pMØ) from Swiss mice were cultured with IB/rCpOsm (1 or 10 µg/ml) in 96-well plates and then infected with L. monocytogenes. IB/rCpOsm (0.1, 1 or 10 mg/kg) was also administered intravenously to Swiss mice, which were then infected intraperitoneally with L. monocytogenes. RESULTS: Pretreatment of the pMØ with IB/rCpOsm significantly increased cell viability after infection and reduced the intracellular bacterial load. The infiltration of neutrophils into the peritoneal cavity of mice pretreated with IB/rCpOsm at 10 mg/kg (but not 0.1 and 1 mg/kg) was reduced after infection. In these mice, the bacterial load was high in the peritoneal fluid and the liver, but histological damage was discrete. The treatments with IB/rCpOsm at 10 mg/kg significantly increased the expression of the anti-inflammatory cytokine IL-10. CONCLUSION: This study shows that recombinant osmotin inclusion bodies from C. procera were bioactive and prompted anti-inflammatory actions at therapeutic dosages in the L. monocytogenes infection model.

Antiinflammatory and antiinfective effect of caffeine in a mouse model of disseminated salmonellosis.

Caffeine has been reported for its antiinflammatory properties by stimulating phagocytosis. In this study, we investigated the antiinflammatory and antiinfective potential of caffeine in murine macrophage cell cultures and Swiss mice infected with virulent Salmonella enterica serotype typhimurium. Peritoneal macrophages (pMØ) were treated with caffeine on 96-well plates for 24 hr and then infected with Salmonella for 4 hr. In another experiment, the pMØ were first infected with the bacterium for 4 hr and then treated with caffeine for 24 hr. In addition, Swiss mice were inoculated, intraperitoneally, with S. typhimurium and then received caffeine intravenously. Control groups received phosphate-buffered saline (PBS) or dexamethasone. We found that treatments with caffeine increased the macrophage cell viability and reduced the intracellular bacterial load. The administration of caffeine to Swiss mice reduced the infiltration of leukocytes into the peritoneal cavity after the bacterial challenge. Furthermore, the bacterial burdens in the peritoneal fluid, bloodstream, spleen, and liver were decreased by caffeine treatment. The expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, and inducible nitric oxide synthase (iNOs) were down-regulated after infection in caffeine-treated mice. We can conclude that caffeine has both antiinflammatory and antiinfective properties that can be useful for management of bacterial infections along with antibiotics.

Anti-infective activity of Cratylia argentea lectin (CFL) against experimental infection with virulent Listeria monocytogenes in Swiss mice.

BACKGROUND: The lectin from Cratylia argentea (CFL) is able to modulate the immune system response and is thus a potential phytotherapeutic substance. HYPOTHESIS/PURPOSE: In this study, we investigated the role of CFL on control of bacterial infection caused by Listeria monocytogenes, the causative agent of human listeriosis. STUDY DESIGN: Swiss mice were infected with L. monocytogenes and then treated with CFL. METHODS: Adult Swiss mice weighing with 30-40 g were infected intraperitoneally with a bacterial suspension (0.2 ml; 1 × 107 CFU/ml). After 30 min, the mice were treated with CFL intravenously at concentrations of 0.1 or 10 mg/kg. Control mice received phosphate-buffered saline (PBS). The animals were euthanized 24 h after infection. RESULTS: We observed that i.v. administration of CFL to Swiss mice did not cause acute toxicity, and reduced the leukocyte counts in the bloodstream 24 h after infection with virulent L. monocytogenes. There was a reduction in the bacterial burden within peritoneal macrophages after infection in CFL-treated mice. Accordingly, the bacterial counts in the bloodstream, spleen and liver also decreased in comparison with the PBS group. Histological damage in the spleen and liver was lower in mice that received CFL treatment. In vitro antimicrobial assays demonstrated that CFL does not inhibit the growth of L. monocytogenes. The mRNA expression of the anti-inflammatory cytokine IL-10 was enhanced with CFL treatment after infection. CONCLUSION: The lectin from C. argentea (CFL) has immunomodulatory and anti-infective properties of pharmacological interest for control of infectious diseases.

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.