Mig Gene Research Articles

CylE and mig as virulence genes of streptococci isolated from mastitis in cows and buffaloes in Egypt

Objective: A study is carried out to investigate the prevalence of mastitis caused by Streptococcus agalactiae and Streptococcus dysgalactiae in cows and buffaloes, their antibiotics susceptibility, minimum inhibitory concentrations (MIC), and molecular investigation of some virulence genes. Samples: A total of 288 milk samples gathered from 72 mastitic animals from households and small-scale dairy farms in Egypt, were analyzed. Procedures: Isolation and identification of S. agalactiae and S. dysgalactiae were performed using conventional techniques. The identified isolates were examined for antimicrobial resistance by disk diffusion assay, MIC by broth microdilution method as well as virulence genes by PCR. Results: Forty-five out of 72 animals show the clinical signs of mastitis. Microbiological evaluation of 288 mastitis milk samples displayed 190 (65.72%) streptococci strains composing of 60% S. agalactiae and 40% S. dysgalactiae strains. The antibiotic susceptibility tests revealed that S. agalactiae strains were resistant to trimethoprim (100%), followed by tetracycline and minocycline (37.7%). Moreover, S. dysgalactiae strains were highly resistant to lincomycin, tetracycline (87.52 each), followed by trimethoprim (81.6) and minocycline (75%). Additionally, the highest MIC with the widest range (1 to ≥128 μg/mL) were observed to trimethoprim for S. agalactiae and to erythromycin and lincomycin for S. dysgalactiae. In contrast, the lowest MIC was detected to penicillin, amoxicillin, cefquinome, and erythromycin for S. agalactiae and to penicillin, amoxicillin, cephapirin, cefquinome, and rifaximin for S. dysgalactiae. The cylE gene is displayed in 6 (60%) S. agalactiae strains, whereas the mig gene is found in 4 (40%) S. dysgalactiae strains.

English

In the present study, 15 S. agalactiae out of 56 streptococcal isolates recovered from 98 milk samples collected from clinical cases, one organized farm and two unorganized sectors in and around Bangalore. All the streptococcal isolates were confirmed at genus level using genus specific primers targeting tuf gene of Streptococcus. Species level identification for S. agalactiae, S. dysgalactiae and S. uberis was done using 16S rRNA. Primers were designed for targeting cfb gene of S. agalactiae, mig gene of S. dysgalactiae, whereas for targeting sip, hyl gene of S. agalactiae and skc, pauA gene of S. uberis either published or designed earlier were used to screen for virulence genes of streptococcal isolates and reference strains. Desired amplicons for the virulence genes were obtained. All the S. agalactiae isolates were also screened for CAMP factor phenotypically by employing CAMP test which was demonstrable in fourteen isolates but cfb gene encoding for CAMP factor was detectable by PCR in all the isolates. The study ultimately helps us to understand the virulence characteristics and mechanisms behind emergence of new strains or shifts in mastitis epidemiology in response to control measures, including antibiotic treatment and vaccination. Key words: Streptococci, virulence factors, bovine mastitis, CAMP factor.

The relative frequency of Mycobacterium tuberculosis and Mycobacterium avium infections in HIV positive patients, Ahvaz, Iran

Objective To estimate the prevalence of Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium avium (M. avium) infections in HIV-positive patients suspected to have pulmonary and extrapulmonary mycobacterial co-infection using PCR technique. Methods Totally 50 samples comprising sputum, pleural fluid and CSF taken from HIV positive patients suspected to have mycobacterial infection, were processed. The demographic information and results of acid fast staining and culture were recorded for each patient. The PCR for detecting of M. tuberculosis comprised of specific primers targeting IS6110 gene sequence. For detecting of M. avium, PCR with primers that amplifies the mig gene were used. Results From 50 samples processed, 45 were sputum (90%), 3 pleural fluid (6%) and 2 CSF (4%). In total, 8 (16%) were culture positive, 7 had positive acid fast staining (14 %) and 13 samples (26%) were positive using PCR technique. All the positive samples were sputum and belonged to patients with pulmonary infection. Of these, 9 were positive for M. tuberculosis (69.2%) and 4 were identified as M. avium (30.8%), which 2 out of 13 positive samples showed mixed infections by both mycobacteria. Conclusions The PCR shows the highest detection rate (26%) of mycobacteria compared with culture and acid fast staining. The majority of infections were with M. tuberculosis (18%) and this shows the importance of this mycobacterial co-infection in HIV positive patients in the region of study.

A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response.

BackgroundThe ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood.MethodsWhole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot.ResultsAntigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 μL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.ConclusionsA monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation is required before processing.

The Mycobacterium tuberculosis homologue of the Mycobacterium avium mig gene is not specifically expressed in the macrophage

With the completion of genome sequencing of Mycobacterium tuberculosisand upsurge in the incidence of M. tuberculosis infection worldwide partly asa result of HIV pandemic, there is need for rationale approach to vaccine andchemotherapy discoveries for M. tuberculosis. The homologue of mig gene ofMycobacterium avium was searched for in the M. tuberculosis database at TheInstitute of Genomic Research (TIGR), USA and The Sanger Institute, UK.Homologue of the gene was found and comprehensively analysed. Reversetranscription PCR (RT-PCR) was carried out on the mig (fadD19) genehomologue and echA19 gene. The result of the RT-PCR showed that the miggene was at least 2-fold upregulated during intracellular infection ofmacrophage compared to the broth grown bacilli as opposed to thedemonstrated specific expression of mig gene in M. avium infectedmacrophage. The echA19 gene was also found to be upregulated. .(Afr. J. Biomed. Res. 11: 173 - 181)Key Words; macrophage induced gene, mig gene, Mycobacterium tuberculosis

Monokine Induced by Interferon-γ (MIG/CXCL9) Is Derived from Both Donor and Recipient Sources during Rejection of Class II Major Histocompatibility Complex Disparate Skin Allografts

Chemokines, including monokine induced by interferon-gamma (Mig/CXCL9), are produced both in allografts and during the direct T-cell infiltration that mediates graft rejection. Neither the specific production nor contribution of allograft donor versus recipient Mig in allograft rejection is currently known. C57BL/6 mice with a targeted deletion in the Mig gene were used as both skin allograft donors and recipients in a class II major histocompatibility complex-mismatched graft model to test the requirement for donor- versus recipient-derived Mig for acute rejection. B6.Mig(-/-) allografts had a 10-day prolonged survival in B6.H-2(bm12) recipients when compared with wild-type C57BL/6 allograft donors, and B6.H-2(bm12) skin allografts had a 5-day prolonged survival in B6.Mig(-/-) versus wild-type recipients. Transplantation of B6.Mig(-/-) skin grafts onto B6.H-2(bm12).Mig(-/-) recipients resulted in further prolonged allograft survival with more than 30% of the grafts surviving longer than 60 days. Prolonged allograft survival was also associated with delayed cellular infiltration into grafts but not with altered T-cell proliferative responses to donor stimulators. Immunohistochemical staining of allograft sections indicated that Mig is produced by both donor- and recipient-derived sources, but Mig from each of these sources appeared in different areas of the allograft tissue. These results therefore demonstrate the synergy of donor- and recipient-derived Mig in promoting T-cell infiltration into allografts.

Effect of Radiation on Gene Expression of Rat Liver Chemokines:In VivoandIn VitroStudies

The aim of the study was to analyze the effect of a single irradiation on chemokine gene expression in the rat liver and in isolated rat hepatocytes. RNA extracted from livers and from hepatocytes within the first 48 h after irradiation was analyzed by real-time PCR and the Northern blot assay. The chemokine concentrations in the serum of irradiated rats were measured quantitatively by ELISA. A significant radiation-induced increase of CINC1, IP10, MCP1, MIP3alpha, MIP3beta, MIG and ITAC gene expression could be detected at the RNA level in the liver. CINC1, MCP1 and IP10 serum levels were significantly increased. In rat hepatocytes in vitro, only MIP3alpha showed a radiation-induced increase in expression, while CINC1, IP10, MIP3beta, MIG, MIP1alpha, ITAC and SDF1 RNA levels were significantly down-regulated. However, incubation of irradiated hepatocytes in vitro with either TNF-alpha, IL1beta, or IL6 plus TNF-alpha led to up-regulation of MCP1, IP10 and MCP1 or CINC1 and MIP3beta, respectively. Irradiation of the liver induces up-regulation of the genes of the main proinflammatory chemokines, probably through the action of locally synthesized proinflammatory cytokines. The reason for the lack of liver inflammation in this model has still to be clarified.

M1 protein of Streptococcus pyogenes increases production of the antibacterial CXC chemokine MIG/CXCL9 in pharyngeal epithelial cells

Streptococcus pyogenes adheres to epithelial cells of the human pharynx where it can cause pharyngitis. To counteract infection, inflamed epithelium produces peptide antibiotics, among them the CXC chemokine MIG/CXCL9. M protein is both a surface-associated and released virulence factor of S. pyogenes. Here, we show that soluble M1 protein enhances MIG gene expression and synthesis in IFN- γ stimulated epithelial cells. M1 protein was recognized both by resting and IFN- γ activated pharyngeal epithelial cells as detected by activation of the transcription factor NF- κB. Furthermore, pharmacological inhibition of NF- κB, decreased MIG synthesis in IFN- γ activated cells, demonstrating a key role for NF- κB in mediating the enhanced response. Microarrays were used to investigate expression of recognized antimicrobial peptides in pharyngeal epithelial cells after stimulation with a combination of IFN- γ and M1 protein. Amongst the most up-regulated and expressed genes, were several antibacterial CC and CXC chemokines. To investigate an in vivo context, pharyngeal mucosa was stimulated in vitro and MIG could be detected by immunohistochemistry in epithelial cells. The results show that epithelial cells can recognize solubilized M1 protein and intact S. pyogenes, thereby modulating an antibacterial innate host response that may have bearing on the outcome of streptococcal pharyngitis.

Monokine Induced by Interferon-γ (MIG) Serum Levels Are a Marker of Disease Load and Correlate with Prognosis in Multiple Myeloma.

Background and Aims: Monokine-induced by interferon-γ (MIG) is a chemokine that is produced by monocytes and macrophages in response to interferon-γ and acts as a chemoattractant mainly to T-lymphocytes in inflammatory processes. MIG has also been suggested to act in an autocrine loop to stimulate tumour cells through its receptor CXCR3, which is known to be expressed in myeloma cells. However, it is presently unclear if MIG is of biologic significance in myeloma in vivo. We have recently shown that multiple myeloma oncogene 1 (MUM1) expression in myeloma cells correlates with prognosis in this disease (Heintel D et al., ASH 2005), and MUM1 was found to upregulate MIG gene expression in B cell malignancies (Uranishi M et al., Leukemia 2005). This led us to evaluate the potential prognostic significance of MIG serum levels in a series of well characterized myeloma patients.Methods: 105 newly diagnosed multiple myeloma patients (median age 69.3 years, range 39.4–90.5) were enrolled. 18 patients presented with Durie/Salmon stage I disease, 9 had stage II and 78 had stage III. MIG serum levels were determined by a commercially available ELISA (R&D Systems). Serum samples from 17 MGUS patients and 37 age-matched healthy volunteers were used as controls.Results: MIG serum levels were elevated in multiple myeloma patients (median 161.3 pg/ml, range 9.37–1966.0) compared to MGUS patients (median 92.7 pg/ml, range 6.29–1303.1) and healthy controls (median 106.2 pg/ml, range 51.0–390.6). For analysis of myeloma cases, a cut-off level for MIG of 200pg/ml (=95th percentile of MIG in controls) was chosen to identify low and high MIG expressers. Using the cut-off as defined above, 63 patients with low MIG serum levels and 42 high MIG expressers were identified in a population of 105 myeloma patients. MIG serum levels in myeloma patients showed strong correlations with several markers of tumour load including low albumin and high β2-microglobulin. Interestingly, no correlation was found with C-reactive protein levels, indicating that MIG is not associated with an inflammatory response in myeloma. Median survival was significantly shorter in patients with high MIG serum levels compared to patients with low MIG expression (median not reached vs. 17.0 months, p<0.001; see figure).Conclusions: MIG serum levels correlate with markers of disease burden in myeloma and high MIG levels are associated with a poor outcome in this disease. [Display omitted]

INFECTIONS CAUSED BY MYCOBACTERIUM AVIUM SUBSPECIES AVIUM, HOMINISSUIS, AND PARATUBERCULOSIS IN FREE-RANGING RED DEER (CERVUS ELAPHUS HIPPELAPHUS) IN AUSTRIA, 2001–2004

Between 2001 and 2004, 14 Austrian free-ranging red deer (Cervus elaphus hippelaphus) infected by Mycobacterium avium species were observed. Eight of the cases were from different geographical regions, and six originated from the same hunting area. The affected animals had signs of diarrhea, severe weight loss, and emaciation. On post-mortem examination, lymphadenitis associated with grossly enlarged mesenteric lymph nodes as well as multiple caseous or purulent nodular lesions in the thickened wall of the intestines were present in all animals. In 10 cases M. avium subsp. avium and in four cases M. a. hominissuis were isolated. In three red deer, a mixed infection with M. a. hominissuis and M. a. paratuberculosis was evident. Typing of M. a. avium and M. a. hominissuis isolates was performed by polymerase chain reaction (PCR) detection of insertion sequence IS901 and the virulence-associated macrophage-induced gene (mig), inverted repeat (IR) typing (IS1245/IS1311), and random amplified polymorph DNA (RAPD) analysis. While all M. a. avium and M. a. hominissuis contained the mig gene, IS901 was detected only in M. a. avium. The prevalence of IS901-positive isolates correlated well with the geographic location of affected animals. The IS901-containing isolates were shown to be genotypically closely related, as they exhibit similar patterns in IR-typing and in RAPD analysis. In contrast, IS901-negative isolates (M. a. hominissuis) displayed distinct profiles in both molecular systems.

Mycobacterium colombiense sp. nov., a novel member of the Mycobacterium avium complex and description of MAC-X as a new ITS genetic variant

Forty-five mycobacterial strains isolated from 23 Colombian HIV-positive patients were identified as members of the Mycobacterium avium complex (MAC) and were characterized using different molecular approaches. Seven of the isolates showed characteristic features that allowed them to be differentiated from other members of the complex. The isolates had a novel 16S-23S rRNA internal transcribed spacer (ITS 1) gene sequence which is described as a new sequevar, MAC-X. All of the seven novel isolates gave a positive result with the MAC-specific AccuProbe (Gen-Probe), but tested negative for Mycobacterium avium and Mycobacterium intracellulare species-specific probes (64 and 100 % of the isolates, respectively). The novel isolates could be differentiated phenotypically from other members of the MAC on the basis of the production of urease and by a consistent mycolic acid pattern. The novel isolates shared some characteristics with M. avium, such as the avium variant I (av-I) pattern of the hsp65 gene as determined by PCR restriction analysis and a positive PCR result for the mig (macrophage-induced) gene. However, the novel isolates showed a unique 16S rRNA gene sequence. DNA-DNA relatedness values, from 24 to 44 %, confirmed the distinction of the novel isolates from other members of the MAC at the genetic level and their status as members of a separate species. The novel isolates are proposed as representatives of a novel species, Mycobacterium colombiense sp. nov., that is closely related to M. avium within the MAC. The type strain is 10B(T) (=CIP 108962(T)=CECT 3035(T)).

Synthesis of α-Chemokines IP-10, I-TAC, and MIG Are Differentially Regulated in Human Corneal Keratocytes

Chemokines responsible for recruiting lymphocytes such as activated T cells into the cornea have not been clearly defined. IP-10, I-TAC, and MIG are chemoattractants for these lymphocytes. The goal of this study was to determine whether human corneal keratocyte (HCKs) in culture synthesize these chemokines in response to proinflammatory mediators. HCKs grown in vitro were stimulated with IL-1alpha, TNF-alpha, or IFN-gamma. Induction of alpha-chemokine gene expression was quantitated by real-time PCR and ELISA. Activation of the transcriptional activator STAT1 by IFN-gamma receptors expressed on HCKs was determined by Western blot analysis. HCKs incubated with TNF-alpha, IL-1alpha, or IFN-gamma resulted in a >2000-fold increase in IP-10 protein secretion by 36 hours after stimulation. In contrast, stimulation with TNF-alpha, IL-1alpha, or IFN-gamma induced levels of MIG and I-TAC that were not significantly greater than constitutive levels. Treatment of HCKs with IFN-gamma activated STAT1 and, in combination with either TNF-alpha or IL-1alpha, enhanced MIG and I-TAC synthesis >20-fold. IP-10 synthesis is induced in HCKs by IL-1alpha, TNF-alpha, and IFN-gamma. In contrast, induction of I-TAC and MIG synthesis in HCKs requires costimulation with IFN-gamma and either IL-1alpha or TNF-alpha. The results suggest therefore, that the upregulation of I-TAC and MIG gene expression at sites of corneal inflammation are more tightly regulated than that of IP-10. A role for differential induction of the three alpha-chemokine genes in corneal inflammatory processes at the eye surface is discussed.

Monokine induced by interferon-γ is induced by receptor activator of nuclear factor κB ligand and is involved in osteoclast adhesion and migration

AbstractBone remodeling is accompanied by the differentiation of osteoclasts from the monocyte/macrophage lineage of hematopoietic cells. The osteoclast differentiation process requires receptor activator of nuclear factor κB (NF-κB) ligand (RANKL), which causes complex changes in the expression of various genes. In a cDNA microarray study to identify genes targeted by RANKL, we found that monokine induced by the interferon-γ (IFN-γ) (MIG) gene was up-regulated in osteoclast precursor cells. The increase in MIG expression by RANKL was confirmed by reverse transcription–polymerase chain reaction and Western blot analysis. RANKL induction of MIG required the activity of NF-κB, whose binding site is present in the MIG promoter. MIG induction by RANKL was also dependent on p38 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 1 (STAT1). RANKL stimulated the phosphorylation of Ser727 of STAT1, which required p38 activity. MIG secreted on RANKL treatment could stimulate the migration and adhesion of osteoclast precursors and osteoclasts that were primed to express CXCR3, the MIG receptor, by macrophage–colony-stimulating factor (M-CSF). Therefore, we provide the first evidence demonstrating that RANKL stimulates the serine phosphorylation of STAT1 through the p38 MAPK pathway, causing MIG gene transcription and secretion, which may have a role in recruiting CXCR3-positive osteoclast precursors and osteoclasts to bone remodeling or inflammatory sites.

Induction of interferon-inducible protein-10 and monokine induced by interferon-gamma from human endothelial cells infected with Influenza A virus.

Primary human umbilical vein endothelial cells (HUVECs) were infected with Influenza virus A/Aichi/2/68 (H3N2) in order to determine the role of endothelial cells in mediating inflammation induced upon virus infection. Structural proteins of the virus and mRNA of the M2 protein were detected in the infected cells, indicating that virus infection had occurred in HUVECs. The Influenza A virus-infected HUVECs showed elevated levels of gene expression of interferon (IFN)-inducible protein (IP)-10 and monokine induced by IFN-gamma (Mig), while heat-, formalin- and diethyl ether-inactivated viruses did not enhance the IP-10 and Mig gene expression. The results thus indicate that infection of live Influenza A virus is responsible for elevation of IP-10 and Mig gene expression. The elevation of IP-10 and Mig gene expression in infected HUVECs was not accompanied by the elevation of IFN-gamma gene expression, indicating that the elevation of IP-10 and Mig gene expression was independent of the IFN-gamma pathway.

Expression of the Mig (CXCL9) gene in murine lung carcinoma cells generated angiogenesis-independent antitumor effects

We examined whether expression of monokine induced by IFN-gamma (Mig, CXCL9) in tumors could produce antitumor effects. Murine lung carcinoma cells (A11) were retrovirally transduced with the murine Mig gene (A11/Mig) and were inoculated into syngeneic mice. Although proliferation in vitro of A11/Mig cells was not different from that of parent cells, the growth in vivo of A11/Mig tumors was significantly retarded compared with that of parent tumors. The antitumor effect was dependent on the amount of Mig produced. We compared the expression level of marker genes of lymphocytes and endothelial cells between parent and A11/Mig tumor masses with reverse transcription-polymerase chain reaction. Expression of CD4, CD8alpha, CD40, CD86, CD28, CD31 and vascular endothelial growth factor was not different between the two tumor groups but expression of CD40 ligand, CD80, NK1.1 and CXCR3 was relatively lower in A11/Mig tumors. Although Mig is a chemotactic factor for activated T and NK cells and an inhibitor for angiogenesis, the present data suggested that production of Mig in tumors did not recruit activated T and NK cells efficiently or suppress angiogenesis. The antitumor effects by Mig could be independent of anti-angiogenesis and recruitment of T and NK cells.

The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo.

Monokine induced by IFN-gamma (Mig; CXC chemokine ligand 9) is an IFN-gamma-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4(+) and CD8(+), and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig(-/-) mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig(-/-) mice showed reductions of 50-75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-gamma induced the expression of mumig in APCs, including CD8 alpha(+) and CD8 alpha(-) dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.

Effect of Interleukin-10 on Lipopolysaccahride/Interferon-γ-Induced ChemokineMigGene Expression

Interferon-γ (IFN-γ) is well known as a potent inducer in monokine induced by IFN-γ (Mig) mRNA expression. Although lipopolysaccharide (LPS) alone is weakly effective on Mig mRNA expression. the stimulation of LPS and IFN-γ (LPS/IFN-γ simultaneously has been shown to synergize to produce a high level of Mig mRNA in mouse peritoneal macrophages. In this study, interleukin-10 (IL-10) was found to suppress the LPS/IFN-γ-induced Mig mRNA expression in cell type- and mouse strain-specific fashion, but IFN-γ alone-induced Mig mRNA was unaffected by IL-10 under identical experimental conditions. The IL-10-mediated suppression of LPS/IFN-γ-stimulated Mig mRNA expression was dependent on the concentration of IL-10, and was prevented when the agent was added 2 hours after LPS/IFN-γ treatment. The suppressive action of IL-10 was dependent on a protein synthesis. However, IL-10 did not reduce the stability of LPS/IFN-γ-induced Mig mRNA. These data may have important implications for a previously unrecognized role for IL-10 as a regulator of synergistic effect of LPS on the IFN-γ-induced expression of the Mig gene in macrophages. (Immune Network 2002;2(1):12-18)

Surface-expressed mig protein protects Streptococcus dysgalactiae against phagocytosis by bovine neutrophils.

The mig gene of Streptococcus dysgalactiae, a major bovine mastitis pathogen, encodes two plasma protein-binding receptors, alpha2-macroglobulin (alpha2-M) and immunoglobulin G (IgG). In this study, the mig gene from one S. dysgalactiae isolate was cloned and expressed in Escherichia coli. The IgG receptor region encoded by mig was conserved in 16 S. dysgalactiae strains. An isogenic mig mutant was constructed by allele replacement mutagenesis of the wild-type gene in S. dysgalactiae. The IgG-binding activity was lost in the mig mutant strain, whereas the alpha2-M receptor activity was still expressed but was detected only in the culture supernatant. In flow cytometry phagocytosis and bacterial-colony-counting bactericidal assays, the wild-type strain was found to be significantly more resistant to phagocytosis and killing by bovine neutrophils (PMNs) than the mig mutant strain when bacteria were preincubated with bovine serum. We therefore speculate that the Mig protein of S. dysgalactiae plays a role in virulence of the bacteria by binding to the plasma protein alpha2-M or IgG and thus preventing phagocytosis by bovine PMNs.

The macrophage-induced gene ( mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase

The macrophage-induced gene ( mig) of Mycobacterium avium has been associated with virulence, but the functions of the gene product were still unknown. Here we have characterized the Mig protein by biochemical methods. A plasmid with a histidine-tagged fusion protein was constructed for expression in Escherichia coli. Mig was detected as a 60 kDa protein after expression and purification of the recombinant gene product. The sequence of the fusion gene and of the parent gene in M. avium were reexamined. This confirmed that the mig gene encodes a 550 amino acid protein (58 kDa) instead of a 295 amino acid protein (30 kDa) as predicted before. The 550 amino acid Mig exhibits a high degree of homology to bacterial acyl-CoA synthetases. Two artificial 30 kDa derivatives of Mig were expressed and purified as histidine-tagged fusion proteins in E. coli. These proteins and the 58.6 kDa histidine-tagged Mig protein were analysed for activity with an acyl-CoA synthetase assay. Among the three investigated proteins, only the 58.6 kDa Mig exhibited detectable activity as an acyl-CoA synthetase (EC 6.2.1.3) with saturated medium-chain fatty acids, unsaturated long-chain fatty acid and some aromatic carbon acids as substrates. Enzymatic activity could be inhibited by 2-hydroxydodecanoic acid, a typical inhibitor of medium-chain acyl-CoA synthetases. We postulate a novel medium-chain acyl-CoA synthetase motif. We have investigated the biochemical properties of Mig and suggest that this enzyme is involved in the metabolism of fatty acid during mycobacterial survival in macrophages.

Gene Expression and Production of the Monokine Induced by IFN-γ (MIG), IFN-Inducible T Cell α Chemoattractant (I-TAC), and IFN-γ-Inducible Protein-10 (IP-10) Chemokines by Human Neutrophils

AbstractMonokine induced by IFN-γ (MIG), IFN-inducible T cell α chemoattractant (I-TAC), and IFN-γ-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that bind to a common receptor, CXCR3, and that are produced by different cell types in response to IFN-γ. We have recently reported that human polymorphonuclear neutrophils (PMN) have the capacity to release IP-10. Herein, we show that PMN also have the ability to produce MIG and to express I-TAC mRNA in response to IFN-γ in combination with either TNF-α or LPS. While IFN-γ, alone or in association with agonists such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression, IFN-α, in combination with TNF-α, LPS, or IL-1β, resulted in a considerable induction of IP-10 release by neutrophils. Furthermore, IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10, and I-TAC mRNA and the extracellular production of MIG and IP-10 in neutrophils stimulated with IFN-γ plus either LPS or TNF-α. Finally, supernatants harvested from stimulated PMN induced migration and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes; these activities were significantly reduced by neutralizing anti-MIG and anti-IP-10 Abs, suggesting that they were mediated by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the ability of neutrophils to produce these chemokines might contribute not only to the progression and evolution of the inflammatory response, but also to the regulation of the immune response.