Vacuolar H+-ATPase is down-regulated by the angiogenesis-inhibitory pigment epithelium-derived factor in metastatic prostate cancer cells.

The Vacuolar H+-ATPases (V-ATPases), a multi-subunits nanomotor present in all eukaryotic cells resides in the endomembranes of exocytotic and endocytotic pathways. Plasmalemmal V-ATPases have been shown to be involved in tumor cell metastasis. Pigment epithelium-derived factor (PEDF), a potent endogenous inhibitor of angiogenesis, is down-regulated in prostate cancer cells. We hypothesized that the transduction of PEDF in prostate cancer cells will down-regulate V-ATPase function; that in turn will decrease the expression of the V-ATPase accessory protein ATP6ap2 and a-subunit isoforms that target V-ATPase to the cell surface. To test these hypotheses, we used the human androgen-sensitive prostate cancer cells LNCaP, and its castration-refractory-derivative CL1 that were engineered to stably co-express the DsRed Express Fluorescent Protein with or without PEDF. To determine if PEDF down-regulates the function of V-ATPase, we measured the rate of proton fluxes (JH+) of the cytosolic and endosome/lysosome compartments. The mRNA levels for subunit-a isoforms and the ATP6ap2 were measured using quantitative reverse transcription-PCR. The results showed that PEDF expression decreased the rate of JH+ in metastatic CL1 cells without affecting JH+ in non-metastatic LNCaP cells, when studying pH(cyt). Interestingly, PEDF did not affect JH+ in endosomes/lysosomes either in metastatic cells or in non-metastatic cells. We also showed that PEDF significantly decreases the levels of a4 isoform and ATP6ap2 in metastatic CL1 cells, without affecting the levels of a4 isoform in the non-metastatic LNCaP cells. These data identify PEDF as a novel regulator of V-ATPase suggesting a new way by which PEDF may inhibit prostate tumor growth.

Imported ornamental fish are colonized with antibiotic-resistant bacteria.

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty-four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.

Co-infection of macrophages modulates interferon gamma and tumor necrosis factor-induced activation against intracellular pathogens.

Co-infection of macrophages (M phi) with Toxoplasma gondii and Mycobacterium avium-intracellulare complex (MAC) has been observed in patients with acquired immunodeficiency syndrome (AIDS). In this study we have demonstrated that co-infected murine M phi respond differently to cytokine stimulation than M phi infected with either of the microorganisms alone. Whereas treatment with interferon gamma (IFN-gamma) activated both single and co-infected groups of M phi to kill T. gondii, treatment with TNF did not influence the rate of MAC growth in co-infected M phi, in contrast with the inhibition of growth observed in MAC-infected M phi. These results suggest that in AIDS patients suffering infection with multiple intracellular pathogens, the ability of cytokines to stimulate microbicidal or static activity in mononuclear phagocytes can be impaired by the presence of more than one of the intracellular organisms.

Recombinant cytokines for controlling mycobacterial infections.

Knowing how mycobacteria exploit host cytokines to survive and which cytokines have important roles in host defense against mycobacteria should allow the use of these molecules in the treatment of mycobacterial infections. Both interleukin 2 and interferon gamma have been used to treat patients with leprosy, and granulocyte-macrophage colony-stimulating factor is presently being administered to AIDS patients infected with Mycobacterium avium.

Recombinant granulocyte-macrophage colony-stimulating factor activates human macrophages to inhibit growth or kill Mycobacterium avium complex.

Organisms belonging to the Mycobacterium avium complex (MAC) are associated with life-threatening bacteremia in patients with the acquired immunodeficiency syndrome (AIDS). As these organisms survive within macrophages, we examined the ability of recombinant human granulocyte-monocyte colony-stimulating factor (GM-CSF) to activate human monocyte-derived macrophages to inhibit the intracellular growth or kill the most mouse-virulent MAC strain in our collection that belongs to serotype 1. While unstimulated cells did not inhibit intracellular growth of MAC, macrophages activated by GM-CSF (10-10(4) U/ml) inhibited or killed up to 58 +/- 5% of the initial inoculum. This activation was dose-dependent, with maximal change occurring with a dose of 100 U/ml after 72 hr exposure. Inhibition or killing was demonstrated if GM-CSF was given both before or after establishment of infection. The combination of GM-CSF (10(2) U/ml) plus TNF (10(2) U/ml) augmented macrophage killing (range, 31 +/- 4%) compared with GM-CSF (10(2) U/ml) alone, but the combination of recombinant human interferon-gamma (IFN gamma) plus GM-CSF resulted in a significant decrease in intracellular inhibition of growth or killing (13.3 +/- 2%) compared with 57.7 +/- 5% obtained with GM-CSF alone. These results indicate that: 1) GM-CSF can activate macrophages to inhibit intracellular growth or kill MAC; 2) killing may be augmented by TNF; and 3) IFN gamma may impair GM-CSF-dependent macrophage activation.

Natural killer cell activity and macrophage-dependent inhibition of growth or killing of Mycobacterium avium complex in a mouse model.

Natural killer (NK) cells from spleens of normal and Mycobacterium avium complex (MAC)-infected C57 black mice (C57 BL/6 bg/+) were examined for their capacity to activate splenic and peritoneal macrophages from beige mice to inhibit or kill intracellular MAC. Peritoneal and splenic macrophages from beige mice were exposed in vitro to NK cells obtained from MAC-infected and uninfected black mice. NK cells from uninfected black mice were also treated in vitro with recombinant interleukin-2 (IL-2) for 48 h before incubation with macrophages. While control macrophages supported intracellular growth of MAC, macrophages exposed to unactivated NK cells inhibited growth of the intracellular bacteria, as determined 4 days after infection. IL-2 stimulated NK cells, and NK cells obtained from MAC-infected animals were able to activate murine macrophages in vitro to inhibit growth or kill 40.0 +/- 5% and 61.3 +/- 6% of the intracellular bacteria, respectively. In other experiments, beige mice (C57 BL/6 bg/bg) were treated intraperitoneally with NK cells obtained from MAC-infected and uninfected C57 black mice. Peritoneal macrophages harvested from beige mice treated with NK cells activated in vitro with IL-2 killed 24.4 +/- 4% of intracellular bacteria by day 4 after infection. Macrophages obtained from animals treated with NK cells harvested from MAC-infected black mice killed 58.8 +/- 7% of intracellular bacteria by 4 days after infection, in contrast with intracellular growth observed in macrophages obtained from untreated animals and from animals treated with Hanks' solution or unactivated NK cells. These crossover studies suggest that NK cells may be important in host defense against MAC.

Relationships between diet-related changes in the gut microbiome and cognitive flexibility.

Western diets are high in fat and sucrose and can influence behavior and gut microbiota. There is growing evidence that altering the microbiome can influence the brain and behavior. This study was designed to determine whether diet-induced changes in the gut microbiota could contribute to alterations in anxiety, memory or cognitive flexibility. Two-month-old, male C57BL/6 mice were randomly assigned high-fat (42% fat, 43% carbohydrate (CHO), high-sucrose (12% fat, 70% CHO (primarily sucrose) or normal chow (13% kcal fat, 62% CHO) diets. Fecal microbiome analysis, step-down latency, novel object and novel location tasks were performed prior to and 2weeks after diet change. Water maze testing for long- and short-term memory and cognitive flexibility was conducted during weeks 5-6 post-diet change. Some similarities in alterations in the microbiome were seen in both the high-fat and high-sucrose diets (e.g., increased Clostridiales), as compared to the normal diet, but the percentage decreases in Bacteroidales were greater in the high-sucrose diet mice. Lactobacillales was only significantly increased in the high-sucrose diet group and Erysipelotrichales was only significantly affected by the high-fat diet. The high-sucrose diet group was significantly impaired in early development of a spatial bias for long-term memory, short-term memory and reversal training, compared to mice on normal diet. An increased focus on the former platform position was seen in both high-sucrose and high-fat groups during the reversal probe trials. There was no significant effect of diet on step-down, exploration or novel recognitions. Higher percentages of Clostridiales and lower expression of Bacteroidales in high-energy diets were related to the poorer cognitive flexibility in the reversal trials. These results suggest that changes in the microbiome may contribute to cognitive changes associated with eating a Western diet.

Cellular and molecular mechanisms of internalization of mycobacteria by host cells.

Mycobacteria are intracellular pathogens capable of invading mononuclear phagocytes, mucosal epithelial cells (including M cells) and Schwann cells. To enter cells, mycobacteria have been shown to interact with several molecules on macrophage and epithelial cell surfaces. This suggests adaptation to the host environment. In this review we address the strategies used by pathogenic mycobacteria to gain access to the intracellular environment.

Mycobacterium avium interaction with macrophages and intestinal epithelial cells.

Mycobacterium avium is an environmental microorganism that is adapted to live both in the environment (mainly in water and soil) and in bird, fish and mammal hosts. In humans, M. avium infection is seen in patients with some sort of immunosuppression, such as patients with chronic lung disease, and Acquired Immunodeficiency Syndrome. More recently, other populations were shown to be at risk to develop M. avium disease. For the majority of time, humans acquire M. avium through the intestinal tract where the bacterium comes in contact with and translocates the intestinal mucosa. M. avium possesses a unique manner to interact with the intestinal mucosa, and, following invasion, can enter and survive within macrophages and monocytes. Although in vitro entry seems to be dependent on binding to the complement receptor, this finding has not been observed in vivo where the bacterium appears to enter macrophages by alternative mechanisms. The bacterium appears to trigger little inflammatory response, and is able to adapt itself to different environments in the host.

Use of liposome preparation to treat mycobacterial infections.

Infections caused by organisms of the genus mycobacteria, such as tuberculosis M. avium disseminated infection in AIDS patients and leprosy, are extremely common around the world. Mycobacteria are intracellular organisms that invade and multiply chiefly within phagocytic cells. Antibiotic resistance among mycobacteria is a growing concern. M. tuberculosis resistant to INH and rifampin are increasing in major urban centers of the developed and in the developing world. M. avium is characteristically resistant to most anti-tuberculosis antibiotics. Furthermore, therapy of mycobacterial infections takes a long time and most of the drugs have potential side effects and toxicity. In addition, mycobacteria is found within cells and antimicrobials need to be able to achieve adequate concentration within the compartment where mycobacteria is located. Liposome preparations, containing antibiotics, have a theoretical advantage in being able to deliver high concentrations of antimicrobials into the infected cell. Studies done thus far, in vitro and in vivo, have confirmed this premise, when comparing drug entrapped in liposomes with free drug. This paper summarizes the results obtained using liposome preparations to treat mycobacterial infections.

Effect of Mycobacterium avium infection on the influx, accumulation, and efflux of KRM-1648 by human macrophages.

KRM-1648 is a new benzoxazinorifamycin with activity in vitro and in vivo against organisms of the Mycobacterium avium complex. We investigated the ability of 14C-KRM-1648 to concentrate within human monocyte-derived macrophages in vitro. KRM-1648 is rapidly taken up by uninfected macrophages, with 90% of the initial concentration added to the monolayer found within macrophages by 1 h and approximately 80% at 2 h. Comparable results were obtained in assays using macrophages that have been infected with an AIDS-related strain of M. avium for 24 h. In contrast, macrophages infected with M. avium for 3 days, showed an impaired ability to concentrate KRM-1648, primarily because of a significant efflux of the antibiotic (intracellular concentration of 86% of the available drug was present within macrophages at 1 h vs. 47% at 2 h). Daily administrations of KRM-1648 to a macrophage monolayer for 3 consecutive days resulted in significant accumulation of the drug within phagocytic cells. Although the efflux was greater in M. avium-infected macrophages than in uninfected cells, consecutive administration of KRM-1648 led to a total intracellular accumulation of drug that exceeded the initial level and appeared to continue to accumulate. The ability of KRM-1648 to rapidly accumulate in human macrophages, including M. avium-infected cells, may explain, in part, the improved therapeutic effectiveness in animal models against M. avium and M. tuberculosis.

Mycobacterium avium resists exposure to the acidic conditions of the stomach.

Organisms of the Mycobacterium avium complex are common pathogens in immunosuppressed patients such as individuals with AIDS. There is evidence that in AIDS patients, the main route for M. avium infection is the gastrointestinal tract. The stomach is a formidable barrier to pathogens and the ability to resist exposure to pH lower than 3 has been shown to be a virulence determinant of enteric pathogens. Incubation of three clinical isolates of M. avium under acidic pH revealed resistance of M. avium grown both to the exponential and stationary phase at pH 2.2 for 2 h. Inhibition of protein synthesis had no effect on the acid tolerance. When the duration of the incubation at pH 2.2 was extended to 24 h, bacteria grown to the stationary phase had a significantly greater tolerance to acid than exponential phase bacteria. M. avium incubated with acid in the presence of water was significantly more resistant to pH 2.2 than M. avium in the presence of buffer. Pre-adaptation in water prior to exposure to acidic conditions was also associated with increased resistance to pH 2.2. Isoosmolarity of Hank's balanced salt solution appears to be responsible for the impaired resistance to acid between 2 and 24 h of incubation. These findings indicate that M. avium is naturally tolerant to pH<3 and that pre-adaptation under conditions similar to the conditions where M. avium is found in the environment results in increased acid resistance.

Isolation of two subpopulations of Mycobacterium avium within human macrophages.

Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS) patients. Human monocyte-derived macrophages were infected with M. avium strain 101 and a quinolone (Bay y 3118) was used at 8 micrograms ml-1, a concentration that kills growing bacteria but fails to eliminate static organisms. Infected monolayers were treated with Bay y 3118 for 4 days and viable bacteria obtained from the lysis of macrophages were used to infect other macrophages without passage in media. The procedure was repeated five times, after which seven different subpopulations that failed to grow within macrophages were identified. While the DNA fingerprinting confirmed that all came from the same strain, three protein profiles were observed. Static subpopulations were not killed by cytokine-stimulated macrophages, in contrast to the replicating subpopulation. Three of the static subpopulation strains were shown to be auxotrophic for glutamic acid or methionine. All seven non-duplicating subpopulation strains grew well in complete 7H10 agar. The importance of a static subpopulation of M. avium within macrophages is presently unknown. It is possible, however, that the non-growing bacteria would persist within macrophages.

Characterization and expression of secA in Mycobacterium avium.

Mycobacterium avium is both a pathogen that infects several hosts such as humans, pigs, and birds, as well as a microorganism that is encountered in environmental sources (soil and water). Protein secretion by the bacterium is likely to influence its ability to overcome adverse and competitive conditions both within or outside the host. Using a combination of cloning and information available in the databank, we characterized the secA gene from M. avium, encoding for a major preprotein translocase subunit associated with the secretion system of prokaryotics. In addition, we cloned the secA promoter sequence in a reporter construct upstream of a promoterless gfp. It was determined that the secA of M. avium shares large homology with the secA of Mycobacterium tuberculosis but not with secA of Mycobacterium leprae. secA expression was determined to be greater at logarithmic growth phase although it was also expressed at low levels during the stationary phase. secA expression was also observed when the bacteria were incubated in water as well as within human monocyte-derived macrophages and in conditions that are associated with biofilm formation. Future evaluation of the sec pathway in M. avium might provide important information about secreted proteins that are required for survival in different environments.

Mycobacterium avium infection of gut mucosa in mice associated with late inflammatory response and intestinal cell necrosis.

Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS). Patients with AIDS appear to acquire M. avium mainly through the gastrointestinal tract. Previous studies have shown that healthy mice given M. avium orally develop disseminated infection after 2-4 weeks. The chief site of M. avium invasion of the intestinal mucosa is the terminal ileum. To learn more about the pathophysiology of M. avium infection of the intestinal mucosa, C57BL/6 bg+ bg+ mice were infected orally with M. avium strain 101 and groups of six mice were killed each week for 8 weeks. The terminal ileum was then prepared for histopathological studies and electron microscopy. A delayed inflammatory response was observed and influx of neutrophils in the Peyer's patches was the only abnormality seen at 1 week. A severe inflammatory response was seen from week 2 to week 5 and necrosis of intestinal villi was observed 6 weeks after infection. These results indicate that invasion and infection of the normal intestine by M. avium results in a severe inflammatory response with segmental necrosis of the intestinal mucosa.

Mechanisms of Mycobacterium avium pathogenesis.

Infections caused by Mycobacterium avium are common in AIDS patients and patients with chronic lung diseases. The bacterium can be acquired both through the intestinal route and respiratory route. M. avium is capable of invading mucosal epithelial cells and translocating across the mucosa. The bacterium can infect macrophages, interfering with several functions of the host cell. The host defense against M. avium is primarily dependent on CD4+ T lymphocytes and natural killer cells. Activated macrophages can inhibit or kill intracellular bacteria by mechanisms that are currently unknown, but M. avium can invade resting macrophages and suppress key aspects of their function by triggering the release of transforming growth factor beta and interleukin 10. Co-infection with HIV-1 appears to be mutually beneficial, with both organisms growing faster.

Naturally occurring antibodies against Mycobacterium avium complex.

Serum obtained from 57 healthy individuals and patients admitted to the hospital owing to diverse pathological causes, as well as serum from seven patients with AIDS and disseminated Mycobacterium avium complex (MAC) infection, were studied to determine the prevalence of IgG and IgM antibodies against surface proteins of M. avium complex. Immunodot assay to detect serum positivity against MAC and Western Blot technique in order to determine the MAC antigens eliciting antibody production were performed. Sera from 89 percent and 81 percent of the non-AIDS population had IgG and IgM antibodies against MAC antigens, respectively. In contrast, 43 percent and 71 percent of the AIDS population had IgG and IgM antibodies against MAC antigens, respectively, in the serum. To define further the antigens recognized by these naturally occurring antibodies, the serum of 14 non-AIDS and four acquired immunodeficiency syndrome (AIDS) patients were studied. Multiple antigens of MAC, with molecular weight ranging from 10 to 95 kilodaltons were recognized by IgG and IgM antibodies present in the sera. The IgM type antibodies were shown to react mainly against 10 kilodalton and 31 kilodalton antigenic protein, while the IgG type antibodies were produced mainly against the 10, 31, and 65 kilodalton proteins. Although the pattern of reaction was consistent between non-AIDS and AIDS populations, IgM antibodies were not detected against the 10 kilodalton protein nor were IgG antibodies detected against the 31 kilodalton protein in the AIDS population.

Effect of ethanol on the interaction between the macrophage and Mycobacterium avium.

Chronic ethanol ingestion predisposes to tuberculosis and bacterial pneumonia. Mycobacterium avium complex organisms cause bacteremia in patients with AIDS. Human macrophages and murine Kupffer cells exposed to ethanol are more permissive towards intracellular growth of M. avium than control mononuclear phagocytes. Ethanol also has been shown to impair the ability of human macrophages and murine Kupffer cells to respond to stimulation with tumor necrosis factor (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF), and to produce cytokines such as interleukin-1, interleukin-6, and TNF when properly stimulated. The impairment is dependent in part on a downregulation in the number of TNF receptors on the macrophage's membrane. Recent evidence suggests that ethanol in nonlethal concentrations induces stress-related proteins in M. avium, leading to the inhibition of intracellular pathways in the macrophage and, consequently, impairing some of its functions. In summary, ethanol acts both on the host and on the mycobacterium in a complex sequence of events that influence the outcome of the infection.

The use of monoclonal antibodies in the diagnosis of partially treated Pseudomonas aeruginosa meningitis: patient report.

The objective of this study was to evaluate the efficacy of mouse anti-pseudomonas monoclonal antibodies to detect small numbers of pathogenic pseudomonas in the spinal fluid of a patient who had been treated with antibiotics for meningitis. Anti-605 monoclonal antibody against Pseudomonas aeruginosa PAC 605, a rough mutant, showed sensitivity similar to that of anti-Fisher 1 monoclonal antibody in detecting P. aeruginosa Fisher 1 antigens in boiled spinal fluid drawn from a patient with a P. aeruginosa perispinal abscess and partially treated meningitis.

Phagocytosis and intracellular killing of Mycobacterium avium complex by human and murine macrophages.

1. Host defenses against Mycobacterium avium complex (MAC) are poorly defined. Peritoneal macrophages from black and beige mice, and cultured human macrophages were infected in vitro with MAC serotype 1 from an AIDS patient, in the presence or absence of normal or convalescent serum. Bacteria:cell ratio was 1:10. Supernatants and macrophage lysates were cultured 6, 24 and 48 h later to determine the uptake and killing by macrophages. Phagocytosis by activated macrophages, obtained from pre-infected and treated mice or stimulated in vitro with endotoxin, was also studied. 2. Neither convalescent serum nor normal serum caused a significant increase in MAC phagocytosis. 3. Unstimulated macrophages from black or beige mice and humans were incapable of killing the intracellular bacteria. Activated macrophages from all sources phagocytized and killed 80 +/- 4% of the initial inoculum after 48 h in culture. 4. These results demonstrate that activated macrophages are required for optimal intracellular killing of serotype 1 MAC.