Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects alveolar macrophages following aerosol transmission. Lung macrophages provide a critical intracellular niche that is required for Mtb to establish infection in the human host. This parasitic relationship is made possible by the capacity of Mtb to block phagosome maturation following entry into the host macrophage, creating an environment that supports bacillary replication. Apoptosis is increasingly understood to play a role in host defense against intracellular pathogens including viruses, fungi, protozoa and bacteria. In the last 15 years an understanding of the role that macrophage apoptosis plays in TB has begun to emerge. Here we review the history and current state of the art of this topic and we offer a model of the macrophage-pathogen interaction that takes into the account the complexities of programmed cell death and the relationship between various death signaling pathways and host defense in TB.
Animals ; Apoptosis/*immunology ; Humans ; Macrophages/*cytology/*microbiology ; Mycobacterium tuberculosis/*immunology ; Tuberculosis, Pulmonary/*immunology

Efforts have been made to accomplish a long term in vitro cultivation of mouse peritoneal macrophage as a host cell for growth of Mycobacterium lepraemurium. Following the inoculation with live or heat-killed Myco. lepraemuriuum of cultured macrophages either on a cover-slip in Leighton tube or in a small petri dish, microscopic observations of acid-fast (AF) stained slide preparation, and total counts of AF bacilli that were released by ultrasonic treatment from the macrophages in small petri dish have been followed in order to present microscopic and quantitative evidence of the actual multiplication of Myco. lepraemurium in cultured mouse peritoneal macrophage. The results are summarized and conclusions are as follows; 1. Successful long term in vitro cultivation of mouse peritoneal macrophage has been accomplished. The growth medium for tissue culture consisted of NCTC 109;50% heat-inactivated calf serum; 40% and beef embryo extract (diluted 1 : 5); 10% and the medium was renewed every 3 to 4 days. The incubation temperature was 37 degrees C; before and at 30 degrees C; after the inoculation with Myco. lepramurium. The CO2 content inside the CO2 humidity incubator for the cultivation of macrophage was kept at 5%. 2. In cultures of macrophage inoculated with live Myco. lepraemurium, clear features of increases in the number of AF bacilli inside individual cell, of elongation of bacill and of increased solidity in AF staining were observed. However, these features were absent in cultlires of macrophage inoculated with heat-killed Myco. lepraemurium. 3. The ultrasonic treatment of macrophage inoculated with 1ive Myco. lepraemurium, and the quantitative assessment of total number of AF bacilli through the course of 6 to 8 weeks after inoculation has provided partial but substantial evidence of actual multiplication of Myco. lepraemurium in cultured macrophages.
Animal ; Female ; Macrophages/microbiology* ; Mice ; Mycobacterium leprae/growth & development* ; Peritoneum/cytology ; Tissue Culture

To investigate cytotoxic secondary metabolites of Micrococcus sp. R21, an actinomycete isolated from a deep-sea sediment (-6 310 m; 142 degrees 19. 9' E, 10 degrees 54. 6' N) of the Western Pacific Ocean, column chromatography was introduced over silica gel, ODS, and Sephadex LH-20. As a result, eight compounds were obtained. By mainly detailed analysis of the NMR data, their structures were elucidated as cyclo(4-hydroxy-L-Pro-L-leu) (1), cyclo(L-Pro-L-Gly) (2), cyclo( L-Pro-L-Ala) (3), cyclo( D-Pro-L-Leu) (4), N-β-acetyltryptamine (5), 2-hydroxybenzoic acid (6), and phenylacetic acid (7). Compound 1 exhibited weak cytotoxic activity against RAW264. 7 cells with IC50 value of 9.1 μmol x L(-1).
Animals ; Biological Factors ; chemistry ; isolation & purification ; metabolism ; pharmacology ; Cell Survival ; drug effects ; Macrophages ; cytology ; drug effects ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Mice ; Micrococcus ; chemistry ; genetics ; isolation & purification ; metabolism ; Molecular Structure ; Phylogeny ; RAW 264.7 Cells ; Seawater ; microbiology ; Secondary Metabolism

BACKGROUNDIsocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.

METHODSMTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.

RESULTSRT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.

CONCLUSIONSMTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

Animals ; Apoptosis ; genetics ; physiology ; Cell Line ; In Situ Nick-End Labeling ; Interferon-gamma ; metabolism ; Isocitrate Lyase ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Microbial Viability ; Microscopy, Fluorescence ; Mycobacterium smegmatis ; enzymology ; genetics ; growth & development ; Mycobacterium tuberculosis ; enzymology ; genetics ; Nitric Oxide ; metabolism ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transformation, Genetic

OBJECTIVETo investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism.

METHODSThe human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting.

RESULTSS. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells.

CONCLUSIONSS. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

Anthracenes ; pharmacology ; Apoptosis ; physiology ; Caspase 3 ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Mitogen-Activated Protein Kinase 8 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 9 ; antagonists & inhibitors ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Signal Transduction ; physiology ; Staphylococcus aureus ; physiology ; U937 Cells ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMV-beta as report gene.

METHODSMurine peritoneal macrophages were infected with SL7207(pCMV-beta) in vitro, then the expression of the beta-gal were detected by X-gal staining or RT-PCR. After mice were orally immunized with SL7207(pCMV-beta), the expression of beta-gal in the lymphoid tissue were tested by RT-PCR, humoral responses were tested by ELISA, splenic lymphocyte proliferation were tested by 3H-TdR incorporation and cytotoxic T lymphocyte reaction were tested by JAM test.

RESULTSThe results indicated that the plasmid pCMV-beta could be delivered by SL7207 into the nucleus of the murine macrophages efficiently and expressed well in vitro; after mice received oral immunizations with attenuated S.typhimurium SL7207 harboring plasmid pCMV-beta mice, the expression of beta-gal could be detected in the spleen, mesenteric lymph nodes and Peyers patches of the mice. Furthermore, the experiments demonstrated that specific humoral immune responses and cell-mediated immune responses were successfully induced in these immunized mice. Compared with the naked DNA vaccination, SL7207 (pCMV-beta) oral immunization were more efficient in inducing cellular immune responses.

CONCLUSIONSAttenuated Salmonella typhimurium SL7207 could be used as DNA delivery vehicle for oral immunization, which have the ability to deliver the antigen-encoding DNA specifically to APC directly for inducing the specific immune response being dominant with cellular immune response.

Animals ; Bacterial Vaccines ; immunology ; Cell Proliferation ; Cytotoxicity, Immunologic ; Female ; Genes, Reporter ; Genetic Vectors ; Macrophages, Peritoneal ; metabolism ; microbiology ; Mice ; Mice, Inbred BALB C ; Mutation ; Salmonella typhimurium ; genetics ; T-Lymphocytes, Cytotoxic ; cytology ; Transfection ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; beta-Galactosidase ; genetics ; immunology ; metabolism