Culture supernatants from lactobacillus plantarum induce necrosis on a human promyelocytic leukemia cell line.

AIMS: Lactic acid bacteria (LAB) are known to have antitumoral properties, although the intrinsic mechanisms responsible for the execution of this activity are poorly understood. Here, we investigated the ability of concentrated supernatants from Lactobacillus plantarum to promote cell death in a human promyelocytic cell line. METHODS AND RESULTS: We aim to test the hypothesis that concentrated supernatants from Lact. plantarum at 5, 50 or 100 µg/ml for 24 h exert cytotoxic effects on HL-60 cells. Accumulation of reactive oxygen species (ROS) was diminished and nuclear staining with Hoechst 33342 and propidium iodide (PI) determined a necrotic induction in a concentrationdependent sequence. Concentrated supernatants did not modify or reduced the activity of caspase-3. The assessment of phosphatidylserine externalization by annexin V/PI double staining led to a necrotic state, but the treatment did not produce a dissipation of mitochondrial membrane potential (ΔΨm), whereas cell cycle analysis revealed that concentrated supernatants failed to significantly enhance the population of HL-60 cells in the hypodiploid (sub-G1) fraction. CONCLUSIONS: Concentrated supernatants from Lact. plantarum are capable of inducing necrosis rather than apoptosis at high doses in a promyelocytic cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: Here, we demonstrate the cytotoxic properties of concentrated supernatants from Lact. plantarum on a tumor cell line, and then, to open the possibility to analyze the chemical composition to elucidate the bioactive molecules.

Acquired resistance to Listeria monocytogenes during a secondary infection in a murine model fed dietary lipids.

OBJECTIVE: The ω-3 polyunsaturated fatty acids can suppress immune system functions. This property may cause adverse effects by impairing host resistance to infection. The present study focused on estimating the impact of different dietary lipids on the immune system of mice after a secondary infection with Listeria monocytogenes. METHODS: BALB/c mice were divided into five dietary groups of olive oil, fish oil, sunflower oil, high-oleic sunflower oil, or low fat that was administered for 8 wk. The mice were immunized with 10(3) colony-forming units. Thirty-eight days later, each mouse was challenged with 10(4) colony-forming units. Mice survival and bacterial clearance from livers and spleens were determined. In addition, cytokine, chemokine, and adhesion molecule productions were quantified from the sera. RESULTS: Survival percentage in mice fed a fish oil diet was 100% and bacterial numbers from spleen were decreased at 72 h. Interleukin-12, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 productions were decreased. Levels of tumor necrosis factor-α and interferon-γ were increased, whereas macrophage inflammatory protein-1α (MIP-1α) production was unaltered. CONCLUSION: Immune defense in mice fed a fish oil diet was improved after secondary exposure, acquiring an adequate resistance. This result could be attributable to an increase of a T-helper type 1 response.

Dietary antioxidants: immunity and host defense.

Natural antioxidants may be defined as molecules that prevent cell damage against free radicals and are critical for maintaining optimum health in both animals and humans. In all living systems, cells require adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive production of reactive oxygen species (ROS) and to prevent damage to the immune cells. During the inflammatory processes, the activation of phagocytes and/or the action of bacterial products with specific receptors are capable of promoting the assembly of the multicomponent flavoprotein NADPH oxidase, which catalyzes the production of high amounts of the superoxide anion radical (O(2)(-)). Under these particular circumstances, neutrophils and macrophages are recognized to produce superoxide free radicals and H(2)O(2), which are essential for defence against phagocytized or invading microbes. In this state, antioxidants are absolutely necessary to regulate the reactions that release free radicals. Antioxidant nutrients commonly included in the diet such as vitamin E, vitamin C, ß-carotene, selenium, copper, iron and zinc improve different immune function exhibiting an important protective role in infections caused by bacteria, viruses or parasites. As a result, dietary antioxidants have been related to modulate the host susceptibility or resistance to infectious pathogens. Overall, numerous studies have suggested that the development of tolerance, and control of inflammation are strongly correlated with specific immune mechanisms that may be altered by an inadequate supply of either macronutrients or micronutrients. Therefore, the present paper will review the effects of dietary antioxidants on immune cell function and the impact on protection against infectious microorganisms.

The antimicrobial peptide cecropin A induces caspase-independent cell death in human promyelocytic leukemia cells.

Most antimicrobial peptides have been shown to have antitumoral activity. Cecropin A, a linear 37-residue antimicrobial polypeptide produced by the cecropia moth, has exhibited cytotoxicity in various human cancer cell lines and inhibitory effects on tumor growth. In this study, we investigated the apoptosis induced by cecropin A in the promyelocytic cell line HL-60. Treatment of cells with cecropin A was characterized by loss of viability in a dose-dependent manner, lactate dehydrogenase (LDH) leakage, and modest attenuation of lysosomal integrity measured by neutral red assay. An increase of reactive oxygen species (ROS) generation, DNA fragmentation, and phosphatidylserine externalization were quantified following cecropin A exposure at a concentration of 30 microM, whereas cecropin A-induced apoptosis was independent of caspase family members, because the activity of caspase-8 and -9 were irrelevant. Nevertheless, caspase-3 activity showed a significant increase at concentrations of 20-40 microM, but a considerable reduction at 50 microM. Flow cytometry analysis revealed a dissipation of the mitochondrial transmembrane potential (Deltapsi(m)), and the accumulation of cells at sub-G1 phase measured by FACS analysis of propidium iodide (PI) stained nuclei suggested induction of apoptosis. Morphological changes measured by Hoechst 33342 or acridine orange/ethidium bromide staining showed nuclear condensation, corroborating the apoptotic action of cecropin A. Overall, these data indicate that cecropin A is able to induce apoptosis in HL-60 cells through a signaling mechanism mediated by ROS, but independently of caspase activation.

Orally administered Lactobacillus plantarum reduces pro-inflammatory interleukin secretion in sera from Listeria monocytogenes infected mice.

Lactic acid bacteria have traditionally been thought to have immunomodulating effects. To verify this property, Lactobacillus plantarum was orally administered to mice (5 x 107 colony forming units (c.f.u.)), prior to infection with Listeria monocytogenes in order to evaluate the host resistance against an infectious micro-organism and to better define the influence of L. plantarum on such responses. Balb/c mice were treated daily with L. plantarum or received PBS (sham-treated mice as controls) for 4 weeks. Subsequently, mice were intravenously infected with a clinical isolate of L. monocytogenes. Our study revealed that the administration of L. plantarum did not significantly increase the survival (P = 0.13) of mice (fifteen in each group) after L. monocytogenes infection (106 c.f.u./ml), whereas a sub-lethal dose of L. monocytogenes (105 c.f.u./ml) was eliminated from liver and spleen 5 d after the challenge in both L. plantarum- and sham-treated mice (n 5). Nevertheless, the levels of IL-1beta and IL-6 from sera of orally administered L. plantarum were drastically reduced at 0, 4 (P < 0.01) and 6 d after L. monocytogenes infection, whereas TNF-alpha production was unaltered. In conclusion, administration of L. plantarum reduced pro-inflammatory IL production after challenge with L. monocytogenes, although it did not significantly impact the survival of mice. We speculate that L. plantarum could exert anti-inflammatory effects, which may represent an important model to reduce inflammatory disorders. Therefore, further studies in human subjects should determine the role of L. plantarum as an immunomodulatory micro-organism and its relationship in the host protection to pathogens.

Significance of olive oil in the host immune resistance to infection.

The effects exerted by polyunsaturated fatty acids (PUFA) on immune system functions have been investigated in recent years. These studies have reported the important role that n-3 PUFA play in the diminution of incidence and severity of inflammatory disorders. Nevertheless, less attention has been paid to the action of monounsaturated fatty acids (MUFA) upon the immune system. The administration of a diet containing a high amount of olive oil in experimental animals produces a suppression of lymphocyte proliferation, an inhibition of cytokine production and a reduction in natural killer (NK) cell activity. Despite these alterations in immune functions, it has been reported that olive oil-rich diets are not as immunosuppressive as fish oil diets. An important aspect in immunonutrition is focused on the relationship between fats, the immune system and host resistance to infection, particularly when these nutrients are supplied to patients at risk of sepsis. Different studies have determined that olive oil-rich diets do not impair the host resistance to infection. Therefore, olive oil constitutes a suitable fat that may be applied in clinical nutrition and administered to critically ill patients. In the present review, we summarize the current knowledge on olive oil and immune system functions, the biological consequences derived from the administration of diets containing olive oil and the impact of olive oil on immune defence.

Examination of host immune resistance against Listeria monocytogenes infection in cyclophosphamide-treated mice after dietary lipid administration.

BACKGROUND & AIMS: Despite the beneficial effects in the resolution of inflammatory disorders due to their immunosuppressive properties, n-3 polyunsaturated fatty acids are associated with a reduction of immune resistance to some microorganisms. Here, we examine the influence of different dietary lipids on host immune resistance against Listeria monocytogenes in mice treated with cyclophosphamide (CPA). METHODS: Balb/c mice were fed one of four diets, which contained either olive oil (OO), fish oil (FO), hydrogenated coconut oil (HCO) or low fat (LF) for 4 weeks. Subsequently, mice were treated with CPA or PBS, prior to L. monocytogenes infection. Splenocyte proliferation, survival analysis, counts of viable bacteria from spleens and livers, and measurement of pro-inflammatory cytokine levels were determined. RESULTS: The FO-rich diet reduced survival, particularly in CPA-treated mice. CPA was responsible for a significant increase of viable bacteria recovery from spleens and livers within each group fed high fat diets, which was aggravated in mice fed an FO diet. In addition, a significant increase of both TNF-alpha and IL-12p70 levels was detected in this group. These results may acquire a crucial relevance in clinical nutrition, particularly when FO diets are administered to immunocompromised patients. CONCLUSIONS: The mechanism(s) that impair(s) the elimination of L. monocytogenes could be associated with a low mitogen-stimulated splenocyte proliferation, and with an alteration of pro-inflammatory cytokine production. The application of the neutropenic agent CPA moderately aggravates the immunosuppressive state mainly in FO-fed animals.

In vitro biological activities of magainin alone or in combination with nisin.

Antimicrobial peptides have received increasing attention not only as potential candidates to their administration as antimicrobial agents, but also as potential drugs applied in cancer therapy. Here, we have examined the action of both nisin and magainin on human promyelocytic leukemia HL-60 cells. Cells were cultured in presence of either nisin or magainin 1 as well as in combination with both nisin and magainin 1. Results have revealed that magainin, but not nisin, produces a loss of cell viability in HL-60 cells, and a minor increase of hemolysis, whereas it is not responsible for cell membrane disruption and lactate dehydrogenase (LDH) leakage. In addition, magainin is involved in a significant generation of reactive oxygen species (ROS), as well as in an augment of caspase-3 activity. Magainin-induced apoptosis was verified by DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining of the cells. Promotion of cell death by magainin occurs via cytochrome c release accompanied by a substantial increase of proteasome activity. These results underline the importance of magainin as a drug capable of exerting an in vitro antitumoral activity by triggering apoptosis.

Assessment of interleukin-12, gamma interferon, and tumor necrosis factor alpha secretion in sera from mice fed with dietary lipids during different stages of Listeria monocytogenes infection.

Recent experimental observations have determined that long-chain n-3 polyunsaturated fatty acids suppress immune functions and are involved in the reduction of infectious disease resistance. BALB/c mice were fed for 4 weeks with one of four diets containing either olive oil (OO), fish oil (FO), hydrogenated coconut oil, or a low fat level. Interleukin-12p70 (IL-12p70), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) production in the sera of mice fed these diets and challenged with Listeria monocytogenes were determined by enzyme-linked immunosorbent assay. In addition, bacterial counts from spleens of mice were carried out at 24, 72, or 96 h of infection. Here, we quantified an initial diminution of production of both IL-12p70 and IFN-gamma, which appear to play an important role in the reduction of host resistance to L. monocytogenes infection. In addition, an efficient elimination of L. monocytogenes was observed in spleens of mice fed a diet containing OO at 96 h of infection, despite reductions in IL-12p70 and TNF-alpha production, suggesting an improvement of immune resistance. Overall, our results indicate that the initial reduction of both IL-12 and IFN-gamma production before L. monocytogenes infection represents the most relevant event that corroborates the impairment of immune resistance by n-3 polyunsaturated fatty acids during the different stages of infection. However, we speculate that the modulation of other cytokines must be also involved in this response, because the alteration of cytokine production in mice fed an FO diet in a late phase of L. monocytogenes infection was similar to that in mice fed OO, whereas the ability to eliminate this bacterium from the spleen was improved in the latter group.

Changes in the immune functions and susceptibility to Listeria monocytogenes infection in mice fed dietary lipids.

The direct examination of the effects that fish oil diets (composed of long-chain n-3 polyunsaturated fatty acids) exert on immune system function indicates a reduction of host natural resistance to infectious diseases mainly because of a suppression of immune function generated by the fatty acids contained in this diet. Here, we evaluated the concentration of IL-12, IL-4, prostaglandin E2 and leukotriene B4 in the serum from BALB/c mice receiving four different diets. Each group was fed a diet that differed only in the source of fat: a low-fat diet (2.5% by weight), an olive oil diet (20% by weight), a fish oil diet (20% by weight) or a hydrogenated coconut oil diet (20% by weight). Mice were fed for 4 weeks and then infected with the intracellular pathogen Listeria monocytogenes. An initial reduction in the Th1-type response as a result of a decrease in IL-12p70 secretion, an inefficient action of IL-4 (Th2-type response) and no modification of pro-inflammatory lipid-mediator production could be, at least in part, the key events responsible for the inadequate elimination of L. monocytogenes from the spleens of mice fed a fish oil diet. Furthermore, our results suggest that the type of dietary lipids may affect the circulating concentration of IL-12p70 and IL-4, leading to a modulation in the protective cellular immune response to L. monocytogenes infection.

Lack of apoptosis in Listeria monocytogenes-infected thymocytes from mice fed with dietary lipids.

The potential action of certain fatty acids has been studied since the early 1970s. Numerous effects on immune system functions have been related to dietary lipid administration; therefore, several of them have been applied in the treatment of inflammatory disorders. Nevertheless, n-3 polyunsaturated fatty acids may affect host resistance to infectious diseases. In addition, several studies have demonstrated that certain fatty acids are involved in apoptosis induction. Here, we have examined the action of different dietary lipids on the promotion of apoptosis in thymocytes from mice fed with dietary lipids and infected with Listeria monocytogenes. Thus, L. monocytogenes promoted an important cytotoxic effect in all of the groups, but it did not increase the percentage of DNA fragmentation. Similarly, an important increase of caspase-3 activity was demonstrated in OO and FO groups, but infection with L. monocytogenes exerted an inhibitory effect. Finally, L. monocytogenes did not modify proteasome activity among groups fed with dietary lipids. On the basis of this preliminary study, we can state that the infection of thymocytes from mice fed with dietary lipids does not promote a synergistic effect in the induction of apoptosis. Hence, these results may partially serve to elucidate the immune mechanisms involved in cells from mice fed with dietary lipids in an infectious process.

Polyunsaturated fatty acids induce cell death in YAC-1 lymphoma by a caspase-3-independent mechanism.

BACKGROUND: The involvement of certain fatty acids in the induction of apoptosis has been established recently. In fact, considerable attention has been given in the past few years to the participation of polyunsaturated fatty acids as substances capable of modulating tumor cell growth. MATERIALS AND METHODS: Fatty acids such as eicosapentaenoic acid (EPA), linolenic acid (LNA), arachidonic acid (AA), linoleic acid (LA), oleic acid (OA) or stearic acid (SA) were added to YAC-1 tumor cells. RESULTS: Incubation of cells with fatty acids revealed a loss of cell viability in a dose-dependent manner. Quantification of DNA fragmentation showed a significant increase particularly in cells treated in the presence of LA, whereas the accumulation of triacylglycerols in the form of cytoplasmic lipid droplets was significantly enhanced in cells cultured with EPA, LNA or AA. The production of reactive oxygen species (ROS) was substantially increased after cell incubation. Nevertheless, the analysis of caspase-3 activity indicated a relevant increase in cells cultured in the presence of LA, OA or SA, but not in cells cultured with EPA, LNA or AA. CONCLUSION: On the basis of these results, we can speculate that long-chain polyunsaturated fatty acids such as EPA and AA as well as LNA induce cell death in YAC-1 lymphoma by an independent mechanism of caspase-3 activation.

Relevance of dietary lipids as modulators of immune functions in cells infected with Listeria monocytogenes.

Nutritional status may have significant importance for the immune system, and particularly, unsaturated fatty acids may serve as modulators of immune functions. Clinical and epidemiological studies have demonstrated that fatty acids are involved in the reduction of the inflammatory processes that occur in diseases characterized by an overactivation of the immune system. At the same time, an increase in susceptibility to infection has also been reported. The importance of immune system modulation by dietary lipids in the presence of an intracellular bacterial pathogen, such as Listeria monocytogenes, was evaluated in the present study. BALB/c mice were divided into four groups which were each fed a low-fat (2.5% by weight) diet, an olive oil (OO; 20% by weight) diet, a fish oil (FO; 20% by weight) diet, or a hydrogenated coconut oil (HCO; 20% by weight) diet for 4 weeks. In each group, lymphocye proliferation was measured, and a reduction in the stimulation index was observed in the FO and HCO groups. Cytotoxicity exerted by L. monocytogenes was increased in the groups fed diets containing OO and FO after 6 h of incubation with the bacterium. An important increase in the production of reactive oxygen species was found in the groups fed the HCO diet after 12 h of incubation with L. monocytogenes. Finally, invasion and adhesion factors were not modified substantially by the action of dietary lipids, although these factors were reduced in cells from mice fed an FO diet. These results underline the importance of several dietary lipids as biological modulators of immune functions and their crucial role in the alteration of host natural resistance.