Expression microarray was employed in this study to investigate whether the ion channels and their regulatory elements encoding genes participate in the immune response to Mycobacterium tuberculosis infection. The results of a virulent strain were compared with those of the clinically isolated strains. The data demonstrate that K(+), Na(+), Ca(2+) and Cl(-) channels and their regulatory elements, such as the G protein, receptor and second messenger, protein kinase and protein phosphatase were involved in the immune reaction. The clinical strain affected more types of ion channels and respective regulatory elements. The data provides clues for further scrutiny into the role of ion channels and related elements in the interaction between Mycobacterium tuberculosis and host macrophage.
Gene Expression Regulation ; Humans ; Ion Channels ; genetics ; Macrophages ; immunology ; microbiology ; Mycobacterium tuberculosis ; pathogenicity ; Regulatory Elements, Transcriptional ; Tuberculosis ; genetics ; immunology ; microbiology

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 grow Mycobacterium leprae in cultured mouse peritoneal macrophages, studies were made on 1) the purification of M. leprae from lepromatous nodules by trypsinization, 2) growth experiment of purified M. leprae in cu1tured macrophages by in vivo infection-in vitro cultivation technique and 3) the observation of pathological changes in sp1eens of mice induced by intraperitoneal inoculation of purified M. leprae. Results are summarized as follows. 1. A simple and effective procedure is described for purification of M. leprae from biopsied nodules of lepromatous leprosy patients by trypsinization and high speed centrifugation. The procedure resulted in a good yie1d of homogeneous preparation of M. leprae with a negligible contamination of tissue debris. 2. Significant decreases were observed in the numbers of acid-fast bacilli in cultured macrophages and of macrophages harboring acid-fast bacilli by the length of intervals between the time of intraperitoneal inoculation of purified M. leprae and the time of initiation of macrophage cultures. 3. Microscopic examination of stained preparations of macrophages cultured by in vivo infection-in vitro cultivation technique indicated that an apparent increase in the number of acid-fast bacilli in the macrophages occurred when the cultures made at 24 hours and 1 week after inoculation were maintained in vitro up to 2 months or more. 4. Pathological changes in the spleens of mice inoculated with purified M. leprae were of mainly degenerative nature in the red pulp. No multiplication of M. leprae was observed in the spleens of mice up to 5 months after inoculation.
Adolescent ; Adult ; Animal ; Cells, Cultured ; Female ; Human ; Leprosy/microbiology* ; Macrophages/microbiology* ; Male ; Mice ; Mycobacterium leprae/growth & development* ; Mycobacterium leprae/isolation & purification ; Peritoneum/microbiology*

OBJECTIVETo investigate the contamination state of Legionella in cooling water samples from different places in Wuxi city and reveal the molecular biological characteristics of Legionella strains.

METHODS112 parallel water samples (500 ml each) were collected from 56 sites in Wuxi city during year 2009 - 2010. The samples were used for Legionella test and quantitative culture. The isolated Legionella strains were used for serotyping, pulsed-field gel electrophoresis (PFGE), sequence-based typing (SBT), and intracellular growth were tested.

RESULTSThe positive proportion of Legionella was 39. 3% (22/56) among all sampling sites. A total of 29 Legionella strains were isolated, and the serotypes include LP1, LP3, LP5 and LP6. LP1 serotype was the major one with a proportion of 65.5% (19/29). 29 Legionella strains got 17 PFGE types. There were 10 SBT types among 10 Legionella strains with different PFGE types. Comparing to LP1 strain (ATCC 33152), WX2011062 (LP6) and WX2011067 (LP5) had strong intracellular growth ability in mouse peritoneal macrophages J774 cell line (the amount of intracellular bacteria on day 0 after infection were (5.5 +/- 1.32) x 10(5), (3.9 +/- 0.60) x 10(5), (7.8 +/- 0.76) x 10(5) CFU/ml, respectively; the amount of intracellular bacteria on day 3 after infection were (58.3 +/- 1.61) x 10(5), (2700.0 +/- 655.74) x 10(5), (3066.7 +/- 208.17) x 10(5) CFU/ml, respectively).

CONCLUSIONThe Legionella contamination existed in cooling water samples from different places in Wuxi city. Legionella strains isolated showed high genetic variation. Some Legionella strains had vigorous intracellular growth ability.

Air Conditioning ; Animals ; Cells, Cultured ; Environmental Microbiology ; Legionella ; genetics ; growth & development ; isolation & purification ; Legionella pneumophila ; growth & development ; isolation & purification ; Macrophages ; microbiology ; Mice ; Serotyping ; Water Microbiology

OBJECTIVETo investigate the apoptosis of J774A.1 cells induced by Leptospira interrogans and the effect of caspase-3, -6 activation on the apoptosis.

METHODSMouse monocyte-macrophage like cell line J774A.1 was infected by L.interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae Lai strain 56601. The apoptosis or necrosis of infected cells was examined by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The activity of caspase-3, -6, and their cleaved substrates PARP and Lamin A/C were measured by fluorometry and Western Blotting, respectively.

RESULTL. interrogans strain Lai was able to induce apoptosis of J774A.1 cells and the maximal apoptotic rate was(48.81+/-5.95)% when microbe: cell ratio was 100: 1. The maximal activities of caspase-3 and -6 in the infected J774A.1 cells were (1453.41+/-36.07) and (618.65+/-39.82) FU, respectively, which were 16.38- and 9.98-fold of those uninfected cells. PARP and Lamin A/C in the infected cells were detected. Caspase-3 and -6 inhibitors remarkably blocked the L. interrogans-induced apoptosis in J774A.1 cells.

CONCLUSIONL. interrogans is able to induce the apoptosis of J774A.1 cells and intracellular caspase-3 and -6 are closely associated with the apoptosis.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Caspase 6 ; metabolism ; Cell Line ; Leptospira interrogans ; pathogenicity ; Macrophages ; enzymology ; microbiology ; pathology ; Mice

Animals ; Female ; Macrophages ; microbiology ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Mycobacteriophages ; physiology ; Mycobacterium tuberculosis ; virology ; Virus Replication

OBJECTIVETo determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species.

METHODSThe expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay.

RESULTSβ1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets.

CONCLUSIONThere is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

Animals ; Blood Platelets ; metabolism ; microbiology ; Calcium Channels ; metabolism ; Cell Line ; HEK293 Cells ; Host-Pathogen Interactions ; Human Umbilical Vein Endothelial Cells ; Humans ; Integrins ; metabolism ; Leptospira ; pathogenicity ; Macrophages ; metabolism ; microbiology ; Mice ; Monocytes ; metabolism ; microbiology ; NIH 3T3 Cells

The role of Mac-1 as a receptor for Bordetella bronchiseptica infection of alveolar macrophages (AMphi) was examined using 6 strains (2 ATCC and 4 pathogenic field isolates) to assess B. bronchiseptica binding, uptake and replication in primary porcine AMphi. All B. bronchiseptica strains were rapidly killed by porcine serum in a dose- and time-dependent manner. However, heat-inactivated porcine serum (HIS) did not demonstrate any bacterial-killing activity, suggesting that complement may have a direct killing activity. All field isolates were more resistant to direct complement-mediated B. bronchiseptica killing. The uptake of B. bronchiseptica into AMphi was inhibited approximately 50% by antiMac-1 monoclonal antibodies in the medium. However, B. bronchiseptica phagocytosed in the presence of serum or HIS was not altered by anti-Mac-1 antibodies although more bacteria were internalized by addition of serum or HIS. These data suggest that Mac-1 is a target for direct uptake of B. bronchiseptica via opsoninindependent binding. The phagocytosed B. bronchiseptica, either via direct or serum-mediated binding, were efficiently killed by AMphi within 10 hr postinfection. This demonstrates that Mac-1-mediated B. bronchiseptica uptake is a bacterial killing pathway not leading to productive infections in AMphi.
Animals ; Antibodies, Bacterial/blood/immunology ; Bordetella bronchiseptica/*immunology ; Macrophage-1 Antigen/*immunology ; Macrophages, Alveolar/*immunology ; Phagocytosis ; Protein Binding ; Swine/*immunology/*microbiology

OBJECTIVE: Soluble triggering receptors expressed by myeloid cells-1 (sTREM-1) and inflammatory cytokine tumor necrosis factor-α (TNF-α) in macrophage cells were stimulated by Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) to investigate the expression of triggering receptors expressed by myeloid cells-1 (TREM-1) and further explore the correlation between TREM-1 and the pathogenesis of periodontitis. METHODS: THP-1 cells (a human monocytic cell line derived from an acute monocytic leukemia patient) were induced to differentiate THP-1 macrophages by phorbol-12-myristate-13-acetate and were injected with 0 (blank control), 0.5, or 1.0 μg·mL⁻¹ Pg-LPS. The THP-1 cells were then grouped in accordance with incubation time, and each group was incubated for 4, 6, 12, or 24 h. The expression of the TREM-1 mRNA in macrophages was detected by real-time quantitative polymerase chain reaction, while the expression of TREM-1 protein was detected by Western blot; the site where TREM-1 protein expression was observed in macrophages was detected by immunofluorescence staining, and the expression of soluble sTREM-1 and TNF-α in cell culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: Compared with the blank control group, the expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in Pg-LPS-stimulated macrophages was significantly upregulated (P<0.05). The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the supernatant of cell culture was higher in the 1.0 μg·mL⁻¹ Pg-LPS group than in the 0.5 μg·mL⁻¹ group; this expression was statistically significant since the 6, 4, and 4 h time point (P<0.05). Cell immunofluorescence staining showed that TREM-1 protein was positive when the THP-1 macrophages was stimulated by Pg-LPS (1.0 μg·mL⁻¹) for 24 h, and the staining sites of TREM-1 were mainly located in the cell membrane of the macrophages (P<0.05). The expression level of TNF-α increased in groups stimulated by Pg-LPS, and the expression level of TNF-α was significantly higher in 1.0 μg·mL⁻¹ Pg-LPS stimulated groups than in 0.5 μg·mL⁻¹ Pg-LPS-stimulated groups since the 6 h time point (P<0.05). The expressions of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in 0.5 μg·mL⁻¹ Pg-LPS-stimulated macrophages were positively correlated with one another (r=1, P<0.05), but no statistically significant correlation was found in the expression of TNF-α. The positive correlation between sTREM-1 and TNF-α expressions was detected when macrophages were stimulated by 1.0 μg·mL⁻¹ Pg-LPS (r=1, P<0.05). CONCLUSIONS The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the culture supernatant in Pg-LPS-stimulated macrophages was significantly upregulated on the basis of the concentration of Pg-LPS; moreover, their upregulation was positively correlated with one another. The expression of TNF-α in the supernatant of cell culture was also upregulated and was positively correlated with the expression of sTREM-1 at the group of high Pg-LPS concentration (1.0 μg·mL⁻¹). Results reveal that TREM-1, which has been realized as a proinflammatory receptor protein, can promote the development of periodontitis by regulating the expression of TNF-α in macrophages.
Adult ; Humans ; Lipopolysaccharides ; Macrophages ; metabolism ; Myeloid Cells ; Periodontitis ; metabolism ; microbiology ; Porphyromonas gingivalis ; pathogenicity ; Triggering Receptor Expressed on Myeloid Cells-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism