Inhibition Of Intracellular Growth Research Articles

Functional Analysis of Genetic Variations in Surfactant Protein D in Mycobacterial Infection and Their Association With Tuberculosis.

Surfactant proteins (SPs)-A and -D are C-type lectins of the collectin family and function in the clearance of infectious particles in the lungs. Some polymorphisms of SPs that give rise to amino acid changes have been found to affect their function. Several SP-A gene polymorphisms have been reported to be associated with respiratory infection diseases, such as tuberculosis (TB). However, the relationship between surfactant proteins D (SP-D) polymorphisms and TB is still unclear. To study the associations between SP-D polymorphisms and TB, the correlations of SP-D polymorphisms with TB were examined in a case–control study, which included 364 patients with TB and 177 control subjects. In addition, we cloned two major SP-D exonic polymorphism C92T (rs721917) and A538G (rs2243639) constructs and used these for in vitro assays. The effects of SP-D polymorphisms on agglutination and other interactions with Mycobacterium bovis bacillus Calmette–Guérin (M. bovis BCG) were evaluated. In comparison with SP-D 92C (amino acid residue 16, Threonine), our results showed that SP-D 92T (amino acid residue 16, Methionine) had a lower binding ability to M. bovis BCG, a lower capacity to inhibit phagocytosis, lesser aggregation, poorer survival of bacillus Calmette–Guérin (BCG)-infected MH-S cells, and less inhibition of intracellular growth of M. bovis BCG. The case–control association study showed that the 92T homozygous genotype was a risk factor for TB. However, a lesser effect was seen for polymorphism A538G. In conclusion, the results of functional and genetic analyses of SP-D variants consistently showed that the SP-D 92T variant increased susceptibility to TB, which further confirmed the role of SP-D in pulmonary innate immunity against mycobacterial infection.

The presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes improved antigen processing and stimulation of T cells.

Dendritic cells (DCs) are potent antigen-presenting cells necessary to establish effective adaptive immune responses. The cytokine environment that exists at the time of DC differentiation may be an important but often ignored determinant in the phenotypic and functional properties of DCs. Interleukin-27 (IL-27) is a unique cytokine that has both inflammatory and immune suppressive activities. Although it can both promote and oppose activity of different T-cell subsets, mostly anti-inflammatory activity has been described toward macrophages and DCs. However, the specific effect of IL-27 during DC differentiation and how that may change the nature of the antigen-presenting cell has not been investigated. In this report, we show that IL-27 treatment during monocyte-derived DC differentiation enhanced the ability to process antigens and stimulate T-cell activity. DCs differentiated in the presence of IL-27 showed enhanced acidification of latex bead-containing phagosomes that was consistent with elevated expression of vacuolar-ATPases. This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the levels of MHC class II surface expression were higher in DCs differentiated in the presence of IL-27. Production of IL-12 was also significantly increased during S.aureus infection of IL-27-differentiated DCs. The net effect of these activities was enhanced CD4(+) T-cell proliferation and T helper type 1 cytokine production. These findings are important to a wide number of immunological contexts and should be considered in the development of future vaccines.

TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.

Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes

Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs. These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9. The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium. We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNγ, IL-1β and LPS. Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides. This results in inhibition of intracellular growth of S. typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN). Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNγ, IL-1β and LPS. These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.

The Lymphocyte Dependent Bactericidal Assay of Human Monocyte and Alveolar Macrophage forMycobacteria

Background : Though mononuclear phagocytes serve as the final effectors in killing intracellular Mycobacterium tuberculosis, the bacilli readily survive in the intracellular environment of resting cells. The mechanisms through which cellular activation results in the intracellular killing is unclear. In this study, we sought to explore an in vitro model of a low-level infection of human mononuclear phagocytes with MAC and and determine the extent of the lymphocyte dependent cytotoxicity of human monocytes and alveolar macrophages. Materials and Methods : The peripheral monocytes were prepared using the Ficoll gradient method from PPD positive healthy people and tuberculosis patients. The alveolar macrophages were prepared from PPD positive healthy people via a bronchoalveolar lavage. The human mononuclear phagocytes were infected at a low infection rate (bacilli:phagocyte 1:10) with MAC(Mycobacterium avium) and Mycobacterium tuberculosis . Non-adherent cells(lymphocyte) were added at a 10:1 ratio. After 1,4, and 7 days culture in , 5% CO2 incubator, the cells were harvested and inoculated in a 7H10/OADC agar plate for the CFU assay. The bacilli were calculated with the CFU/ of the cells and the cytotoxicity was expressed as the log killing ratio. Results : The intracellular killing of MAC and within the monocyte was greater in patients with tuberculosis compared to the PPD positive controls (p within the alveolar macrophage appeared to be greater than that within the monocytes of the PPD positive controls. There was significant lymphocyte dependent inhibition of intracellular growth of the mycobacteria within the monocytes in both the controls and tuberculosis patients and within the macrophages in the controls(p. Conclusion : This study is an in vitro model of a low-level infection with MAC and of human mononuclear phagocytes. The intracellular cytotoxicity of the mycobacteria within the phagocytic cells was significantly lymphocyte dependent. During the 7 days culture after the intracellular phagocytosis, the actual confinement of the mycobacteria was observed within the monocytes of tuberculosis patients and the alveolar macrophages of the controls as in the case of adding lymphocytes.

Human platelet inhibition of Toxoplasma gondii growth.

The human platelet contribution against the intracellular growth of the parasite in vitro in human pulmonary fibroblasts was explored. It was observed that tachyzoites of Toxoplasma gondii induced activation of human platelets and additionally that platelets mediated inhibition of intracellular growth in a virulent T. gondii strain. A prominent role for platelet-derived growth factor (PDGF) was demonstrated in this phenomenon, by testing human recombinant PDGF-AA, -AB and -BB and antibodies to human PDGF-AB that partially reversed its effects. Moreover, the effect of PDGF was significantly higher if the host cells were treated 2 h before parasite infection. PDGF was not directly 'toxic' to free tachyzoites, but only affected parasites within host cells. PDGF-mediated inhibition may involve the cyclooxygenase cycle of the fibroblasts being partially reversed by the cyclooxygenase inhibitors, acetylsalicylic acid and indomethacin. However, a thromboxane synthetase pathway was not implicated. PDGF action against intracellular tachyzoites may also include increased IL-6 production in fibroblasts. Finally, transforming growth factor-beta 1 (TGF-beta1), another component of alpha-granules released at the same time as PDGF, may not be antagonistic to the PDGF parasite inhibitory effect in confluent host cells.

Application of Flow Cytometry and Microscopical Methods to Characterize the Effect of Herbal Drugs on Leishmania Spp.

Plock, A., Sokolowska-Köhler, W., and Presber, W. 2001. Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania spp. Experimental Parasitology97, 141–153. The World Health Organization has identified leishmania sis as a major public health problem, particularly in Africa, Asia, and Latin America. About 1.5 to 2 million people are affected annually by this parasitic infection. As there is no vaccine, there is still a strong need for sufficient drugs. In a preliminary screening, extracts of 50 different plants were evaluated for their possible leishmanicidal activity against the promastigote form of Leishmania mexicana amazonensis. Eighteen extracts showed at least 50% inhibition at 100 μg/ml. The ethanolic extract from Yucca filamentosa L. showed the strongest leishmanicidal activity (100% inhibition at 5 μg/ml). The bioactivity-guided fractionation of this extract led to the isolation of three main components (Yuccasaponins MC 1–3). In further experiments, the effect of Yuccasaponin MC 3 on the promastigote form of L. mexicana amazonensis was quantified and characterized using flow cytometry and specific fluorescent dyes [propidium iodide, Syto 9, and DiBAC4(3)]. The data revealed that the membrane of the promastigote is attacked. The effect of Yuccasaponin MC 3 on intracellular forms (amastigote) was also characterized; green fluorescent protein-transfected Leishmania major were used. By this method, an inhibition of intracellular growth of L. major was demonstrated. This paper shows that, together, flow cytometry and microscopy are quick, sensitive, and easily reproducible methods to describe the effects of drugs on parasites.

Construction and characterization of a Mycobacterium tuberculosis mutant lacking the alternate sigma factor gene, sigF.

The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis. The growth rate of the DeltasigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the DeltasigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the DeltasigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the DeltasigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

Induction of Apoptosis in Murine Macrophages byMycobacterium tuberculosisIs Reactive Oxygen Intermediates-Independent

Infection withMycobacterium tuberculosisinduces apoptosis in murine macrophage lines. Resistant macrophages B10R (Bcgr) are more prone to undergo apoptosis than susceptible B10S (Bcgs) macrophages. Apoptosis and inhibition of intracellular growth of the mycobacteria seem to be dependent on the production of nitric oxide, since both can be reverted by aminoguanidine (AMG). Although B10R macrophages produce more superoxide anion than B10S macrophages after infection withM. tuberculosis,reactive oxygen intermediate (ROIs) scavengers did not affect uptake of3H-uracil incorporation by the mycobacteria nor the induction of apoptosis. These results further suggest that both phenomena are dependent on the production of nitric oxide by the infected macrophages.

Simplified quantitative assay system for measuring activities of drugs against intracellular Legionella pneumophila.

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-alpha agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-alpha broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.

Growth within macrophages increases the efficiency of Mycobacterium avium in invading other macrophages by a complement receptor-independent pathway.

Infections caused by organisms of the Mycobacterium avium complex occur in approximately 50 to 60% of patients with AIDS. M. avium is an intracellular pathogen that survives and multiplies within mononuclear phagocytes. In this study, we investigated the uptake of M. avium grown within macrophages (intracellular growth M. avium [IG]) by a second macrophage compared with M. avium cultured in broth (extracellular growth M. avium [EG]). The results showed that IG was six- to eightfold more efficient than EG in entering macrophages. In addition, while an anti-CR3 antibody was able to inhibit approximately 60% of EG uptake by macrophages, it failed to inhibit the entry of IG. In contrast to EG, IG uptake into macrophages was significantly inhibited in the presence of anti-beta1-integrin and anti-transferrin receptor antibodies. Entry into macrophages by alternate receptors was associated with resistance to tumor necrosis factor alpha (TNF-alpha) stimulation. While stimulation with TNF-alpha resulted in inhibition of the growth of EG, it was not associated with inhibition of intracellular growth of IG. Investigation of the reason why M. avium is able to sense the changes in the intracellular environment triggering a change to the invasive phenotype suggests a direct relationship with macrophage apoptosis. These results suggest that intracellular growth is associated with novel mechanisms of M. avium uptake of macrophages and that those mechanisms appear to offer advantages to the bacteria in escaping the host defense.

Excellent Activity of Newer Quinolones on Legionella pneumophila in J774 Macrophages

The activity of six antibiotics directed against intracellularly multiplying Legionella pneumophilia was examined in tissue cultures with J774 macrophages. The drugs tested were the new quinolones, BAY Y 3118 and clinafloxacin, and ciprofloxacin, erythromycin, gentamicin and ampicillin served as reference drugs. Additionally, the MICs of these drugs against L. pneumophila were determined in vitro by broth microdilution. Despite their low MIC values, ampicillin and gentamicin did not inhibit intracellular multiplication of L. pneumophila in J774 macrophages. In contrast, an inhibition of intracellular growth could be demonstrated for the four other antibiotics. The new quinolones BAY Y 3118 and clinafloxacin showed the highest activity against intracellular L. pneumophila. At a concentration of 0.00078 mg/L already, a marked reduction in bacterial counts was seen for both drugs in comparison to the growth control without antibiotics. The corresponding effective concentrations were 0.0125 mg/L for ciprofloxacin and 0.2 mg/L for erythromycin. It may be concluded that new quinolone derivatives might become an alternative to erythromycin and rifampicin which at present are the drugs of primary choice for the treatment of legionnaires' disease.

Marked inhibition of the intracellular multiplication of Legionella pneumophila in monocytes isolated from carriers of human T lymphotropic virus type I

Intracellular growth patterns of Legionella pneumophila were examined in monocytes obtained from carriers of human T-lymphotropic virus type I (HTLV-1) and controls who were HTLV-1 seronegative. All subjects were seronegative for antibodies against L. pneumophila. Bacterial growth was determined 0, 1, 2, and/or 3 days after infecting peripheral blood mononuclear cells (PBMCs) with the bacteria. The intracellular growth of L. pneumophila was markedly inhibited in HTLV-1 carriers compared with normal controls. When the lymphocytes were depleted from the HTLV-1 carrier PBMC cultures before infection, this inhibition was abolished. Inhibition reappeared, however, when the 72-h culture supernatants of PBMCs from HTLV-1 carriers were added to the lymphocyte-depleted cultures. Culture supernatants of infected and uninfected PBMCs from HTLV-1 carriers exhibited markedly increased levels of interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) compared with the HTLV-1 seronegative controls. In the HTLV-1 carriers, IFN-gamma was produced by the CD4+ lymphocytes, whereas TNF-alpha was secreted by the monocytes. Addition of anti-IFN-gamma or anti-TNF-alpha antibodies to the HTLV-1 carrier PBMC cultures diminished the inhibition of intracellular growth of L. pneumophila. Results suggest that the monocytes are activated in HTLV-1 carriers. These findings may explain why an opportunistic infection by certain intracellular pathogens is rarely seen among HTLV-1 carriers.

Inhibition of Intracellular Growth of Staphylococcus aureus by Exposure of Infected Human Monocytes to Clarithromycin and Azithromycin

The direct effect of clarithromycin and azithromycin on human polymorphonuclear leukocyte (PMN) functions and their intracellular activity against Staphylococcus aureus, phagocy-tosed by human monocytes, were studied. The presence of both antibiotics, in the range of concentrations from 0.25 to 20 μ/ml, did not affect chemotaxis, opsonized-zymosan phagocytosis, respiratory burst measured by nitroblue tetrazolium reduction and phorbol myristate acetate-induced superoxide production, or the microbicidal activity of human PMNs against Candida albicans. Both macrolides were bactericidal against staphylococci in the monocyte system, while bacteriostatic activity was found in cell free system. At concentrations equal to the minimum inhibitory concentrations (MICs) (0.75 and 0.1 respectively for azithromycin and clarithromycin) more than 99% of intraphagocytic S. aureus were killed after 24h incubation. Increasing the concentrations of each drug above the MICs (5 and 10 MICs) did not alter the killing rate of intracellular bacteria. Moreover, no differences between the intracellular bioactivity of these antibiotics were demonstrated, despite their different uptake kinetics.

In vitro activity of the benzoxazinorifamycin KRM-1648 against drug-susceptible and multidrug-resistant tubercle bacilli.

We investigated the activity of benzoxazinorifamycin (KRM-1648) against several drug-susceptible and multidrug-resistant strains of tubercle bacilli. Since KRM-1648 is a rifamycin derivative, we included some strains of Mycobacterium tuberculosis resistant to rifampin (RIF) among the multidrug-resistant strains. For RIF-susceptible strains, the MIC of KRM-1648 was much lower than that of RIF (MICs of KRM-1648 and RIF at which 90% of strains are inhibited, < or = 0.015 and < or = 0.25 micrograms/ml, respectively). The MBC of KRM-1648 (range, 0.007 to 0.03 microgram/ml) was also much lower than that of RIF (range, 0.5 to 1.0 microgram/ml). Postantibiotic effect studies with KRM-1648 showed a rapid reduction in the CFU counts with an exposure of 24 h or more, and its sterilizing effect was maintained even up to 21 days thereafter. Parallel postantibiotic effect studies with RIF showed a less significant effect with a faster recovery of growth, and RIF failed to sterilize the organisms even after 72 h of exposure. KRM-1648 at 0.125 and 0.25 microgram/ml caused complete inhibition of intracellular growth of M. tuberculosis in J774 A.1 macrophages after 48 h of exposure. After a similar exposure time RIF at a concentration of 0.25 microgram/ml caused complete inhibition of growth, but a concentration of 0.125 microgram/ml caused only a 50% reduction in growth compared with that of controls at day 7. With 24 h of pulsed exposure of the intracellular organisms to 0.25 micrograms of the drugs per ml, KRM-1648 caused complete inhibition of intracellular growth, while RIF caused only moderate inhibition of intracellular growth. These findings suggest that KRM-1648 is a potentially useful drug for the treatment of tuberculosis.

LPS inhibits the intracellular growth of Legionella pneumophila in thioglycolate elicited murine peritoneal macrophages by iron-dependent, tryptophan-independent, oxygen-independent, and arginine-independent mechanisms.

Thioglycolate-elicited murine macrophages from genetically susceptible A/J mice activated with lipopolysaccharide (LPS) and infected with Legionella pneumophila in vitro evince marked inhibition of intracellular growth of this bacterium. The mechanism of inhibition by LPS-activated macrophages in terms of replication of this intracellular pathogen is unclear. LPS activation of murine macrophages induced a downshift in transferrin receptor (TfR) expression and reduction in cellular iron content, and this was correlated with augmented intracellular growth of Legionella in the cells. When LPS-stimulated macrophages were first saturated with iron, partial reversion of L. pneumophila growth restriction was observed. However, an excess of exogenous L-tryptophan (Trp) did not reverse this growth inhibition, nor did supplementation of the macrophage culture medium with both iron and Trp. The antilegionella activity of the macrophages induced by LPS activation was independent of reactive oxygen intermediates (ROI), since the scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on growth restriction. Likewise, notwithstanding the ability of LPS-activated macrophages to synthesize reactive nitrogen intermediates (RNI), which was inhibited by L-arginine analogs NG-monomethyl-L-arginine and L-aminoguanidine), as well as by incubation in arginine-free medium, their ability to inhibit the intracellular replication of L. pneumophila was not affected. Thus, we conclude that LPS-activated macrophages inhibit the intracellular growth of L. pneumophila partially by iron-dependent, Trp-independent, and ROI- and RNI-independent mechanisms. We also suggest that additional unknown mechanisms are involved, since complete reversion was not obtained.

Effects of clarithromycin and rifabutin alone and in combination on intracellular and extracellular replication of Mycobacterium avium.

The combined effect of clarithromycin and rifabutin against Mycobacterium avium multiplying either within human monocyte-derived macrophages or extracellularly in a liquid medium was additive: both MICs and MBCs were twofold lower for the combination than they were for each drug alone. Prolonged exposure for 4 weeks of M. avium-infected macrophages to combined concentrations that were only twofold greater than the MICs resulted in a 100-fold decrease in the number of viable bacteria, while in the drug-free controls a 100-fold or greater increase in comparison with the initial viable counts took place. Comparison of this effect with the results of the prolonged exposure to each drug alone suggested that under these experimental conditions rifabutin enhanced the antimicrobial activity of clarithromycin against intracellular bacteria. At the same time, inhibition of intracellular growth by a 2-h pulsed exposure of the infected macrophages to the combination of the two drugs was not different from the effect induced by clarithromycin alone. In conclusion, clarithromycin played the major role in the antimicrobial activity of the tested combination, while rifabutin may have enhanced this effect during a prolonged exposure of the intracellular bacteria to these two agents.

Inhibition of intracellular growth of Mycobacterium avium by one pulsed exposure of infected macrophages to clarithromycin.

A single 2-h pulsed exposure of either human monocyte-derived macrophages or J774 cells infected with Mycobacterium avium to clarithromycin at 3.0 micrograms/ml completely inhibited the intracellular bacterial growth during the first four days of observation, and then only a slight increase in the number of CFU per milliliter took place between the fourth and seventh days. These data suggest that in vivo the intracellular bacteria can be effectively inhibited after a short period when the concentration of the drug in blood reaches its maximum. On the basis of these data, the assumptions that the elimination of bacteremia observed in clarithromycin clinical trials is a result of the activity of the drug not only against bacteria in blood but in macrophages as well and that the peak concentration attainable in blood is essential for these effects can be made.

Effects of cytokines on intracellular growth of Brucella abortus

Interleukin 1 alpha (IL-1 alpha), IL-2, IL-4, IL-6, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocyte macrophage colony-stimulating factor (GM-CSF) were tested for their abilities to alter the growth of Brucella abortus in BALB/c J774A.1 murine macrophages. IL-1 alpha, IL-4, IL-6, tumor necrosis factor alpha, and granulocyte macrophage-colony-stimulating factor had no consistent or significant effect on the growth of the avirulent B. abortus strain 19. In contrast, the addition of either IFN-gamma or IL-2 at 100 U/ml to the macrophage cultures resulted in a significant reduction in the number of intracellular bacteria that was not attributable to decreased infection rates. With IL-2, the reduction was most often apparent only during the first 24 h after infection, while inhibition with IFN-gamma was apparent throughout the culture period of 48 h. The addition of either IL-2 or IFN-gamma to macrophage cultures also resulted in reduced intracellular CFU of the virulent B. abortus strain 2308 and the attenuated rough mutant B. abortus strain RB51. Inhibition of intracellular growth was not augmented by combinations of cytokines. Additional studies with IFN-gamma and IL-2 indicated that they could mediate the inhibition of intracellular growth of B. abortus in resident and thioglycolate broth-induced BALB/c peritoneal macrophages and in splenic macrophages. IFN-gamma also inhibited bacterial growth when added after infection of the macrophages, although the magnitude of the antibrucellae effects was less than that when it was added before infection. Furthermore, the maximal inhibitory effect was sustained only when IFN-gamma remained in the cultures after infection of the macrophages.

In vitro activity of nonoxynol 9 on HeLa 229 cells and primary monkey cervical epithelial cells infected with Chlamydia trachomatis.

Nonoxynol 9 (non-9) is the active ingredient in a wide variety of vaginal contraceptive preparations. The manufacturer recommendation for optimal contraceptive practice is repeated application every 6 h. We studied the in vitro activity of non-9 against Chlamydia trachomatis (E/UW-5/Cx) and its toxicity against HeLa 229 cells and monkey cervical epithelial cells. With a contact time of 6 h, non-9 was toxic to HeLa cells at concentrations of 50 micrograms/ml or greater and to monkey cervical cells at 100 micrograms/ml or greater. Inhibitory effects of non-9 on extracellular chlamydiae were observed at concentrations of 50 micrograms/ml or greater. Inhibition of intracellular growth of chlamydiae in monkey cervical cells was observed at a nontoxic concentration of 50 micrograms/ml. Our studies show that non-9 has antichlamydial activity. However, owing to its toxicity to cervical cells in vitro, the effects of prolonged use of non-9 in vivo should be further studied.