1 Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan
2 Department of Molecular Immunology, Nara Institute of Science and Technology, Ikoma, Nara 631-0101, Japan
Correspondence to: T. Seya, Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan
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Abstract |
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Keywords: comparative immunology, cytokines, guinea pigs
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Introduction |
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IL-12 is an early proinflammatory cytokine produced predominantly by macrophages and other antigen-presenting cells (APC) (1,2). IL-12 promotes the differentiation of naive T cells into Th1 cells, induction of IFN- by acting on T and NK cells, and suppression of IgE production, which are essential for eliciting optimal Th1 responses and, hence, cell-mediated immunity. IL-12 also potentiates NK-mediated and cytotoxic T lymphocyte (CTL)-dependent cytolysis. All these functions of IL-12 were confirmed by not only in vitro studies in both human and murine systems, but also in vivo mouse models including IL-12-knockout mice (3), which reinforced the importance of IL-12 in an early stage of establishment of a Th1-dominant state followed by a variety of cellular responses in infectious, allergic and tumor immunity.
Human rIL-12 failed to express the reported immune modulatory activity toward murine lymphocytes because of its species specificity governed by the p35 subunit (4,5), while it exerted these activities toward non-human primates with a wide dose range (6,7). Thus, its activity could not be evaluated in mice, with either ex vivo or in vivo challenge. Moreover, some structural and functional discrepancies between human and mouse systems have been reported. In humans, two subunits (ß1 and ß2) of the IL-12R are needed to facilitate sufficient binding of human IL-12 to the receptor. In contrast, in mice, the ß1 subunit may be sufficient to sustain mouse IL-12 binding, while the ß2 subunit is necessary for signal transduction (1,810). Likewise, binding affinity of human antagonistic p40 homodimer to the receptor is pretty low compared to that of mouse p40 homodimer that competes with the p70 with similar binding affinity to the receptor (1113). These previously reported results indicated that murine IL-12 is not a simple functional substitute for human IL-12. Therefore, it is necessary to characterize other potential small animal models for in vivo analysis of IL-12.
It has been reported that hormonally and immunologically, guinea pigs are more similar to humans than are rodents (14). Particularly, the guinea pig model of tuberculosis (TB) has certain advantages over the mouse model because of similar immune responses with humans, like generating potent skin-test reactions (delayed-type hypersensitivity) and developing substantial necrosis in the lungs after TB exposure, which eventually causes sufficient damage to the lung architecture to be fatal (1416). Protective immunity to TB is mediated by Th1 cells and triggered by the innate immune system including IL-12 (16,17).
Here, we cloned cDNAs of the two subunits of guinea pig IL-12, and compared its predicted structure and function with human, mouse and other species counterparts. Our findings demonstrated that guinea pig IL-12 showed the highest similarity to humans and, unlike mouse IL-12 p35, guinea pig p35 has no ability to define its species-specific functional expression. In addition, IL-12 (both p35 and p40) mRNA, p40 protein and a 200-kDa molecule antigenically related to the p40 subunit were constitutively expressed in the guinea pig testis.
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Methods |
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Preparation of cells and mitogenic stimulation
Guinea pig spleen cells were harvested from minced tissue and dispersed through mesh screens. Erythrocytes were lysed with TrisNH4Cl and the cells were washed 3 times in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 10% FCS. Peritoneal macrophages elicited by thioglycollate medium (TGC) were isolated as described (18). Cells (1x107 cells/ml) were stimulated with 10 µg/ml of lipopolysaccharide (LPS; Escherichia coli 0127: B8; Difco, Detroit, MI) or 5 µg/ml of concanavalin A (Con A; Honen, Tokyo, Japan) in a humidified 5% CO2 incubator for 1224 h at 37°C.
RNA extraction
Total RNAs from the harvested cells or frozen guinea pig tissues were isolated using Trizol (Life Technologies) according to the manufacturer's instructions. mRNA was prepared by oligo(dT) priming of total RNA isolated from LPS-stimulated peritoneal macrophages or testis using a mRNA purification kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Cloning and sequencing of guinea pig IL-12 p35 and p40 subunits
First, guinea pig IL-12 p35 and p40 partial cDNAs were generated using the standard RT-PCR method. Briefly, first-strand cDNAs were reverse-transcribed from random-primed RNA templates prepared from LPS-stimulated peritoneal macrophages using Superscript II reverse transcriptase (Life Technologies). Typical nested-PCR was performed with degenerate primers synthesized according to the conserved amino acid sequences between human and mouse counterparts (Table 1. Second, full-length IL-12 p35, p40 cDNAs were generated using a Marathon cDNA amplification kit (Clontech, Palo Alto, CA). In this process, PCR from a macrophage cDNA template was performed using p35, p40 gene-specific internal primers (Table 1
) paired with either the AP1- or the AP2-specific end primers. The PCR products were ligated directly into the pCR2.1 TA cloning vector (Invitrogen, Carlsbad, CA). In each experiment, eight to 10 clones were sequenced across both strands using a model 373A automated DNA sequencer (PE Applied Biosystems, Foster City, CA).
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Preparation of other cytokines and ß-actin cDNA using RT-PCR
Since human IL-12 p35, p40 and guinea pig IFN- sequences are present in the GenBank data base (accession nos M65271, M65272 and AF058395 respectively), we synthesized appropriate primers and constructed open reading frames (ORF) or partial cDNAs using the standard RT-PCR method. ß-Actin was used as a control housekeeping gene. The primer sets used for the detection of guinea pig ß-actin were those reported by Rottman et al. (19). RNA templates for cloning of human IL-12 p35 and p40 ORF cDNAs were isolated from 12 h LPS (10 µg/ml)-stimulated human monocytes as described below. Clones were characterized and sequenced as described above.
Northern blotting analysis
Total RNA (10 µg) was resolved by electrophoresis on agarose gels containing formaldehyde, transferred onto Hybond N+ membranes (Amersham Pharmacia Biotech) and hybridized with 32P-labeled cytokine cDNA probes by the standard method. The blots were rehybridized with a ß-actin probe to estimate the relative amounts of RNA loaded. The mRNA level was estimated from radioactivity of the hybridized probe by phosphoimaging or autoradiography. For IL-12 probes, the membranes were first probed with the p35 probe, stripped and reprobed with the p40 cDNA probe. Exposure times on Hyper film MP (Amersham Pharmacia Biotech) were 5 days (p35) and 7 days (p40) at 80°C with an intensifying screen. The IFN--probed filters were exposed for 4 days and each of the ß-actin-probed filters were exposed for 1 day at 80°C.
Purification of E. coli-expressed p40 and immunization
Predicted guinea pig mature p40 (amino acids 23332, Fig. 1b) cDNA was ligated into the E. coli expression vector pET-28b (Novagen, Madison, WI), and used to transform competent E. coli BL21 (DE3) cells (Novagen). N-terminal (His)6-tagged p40 was purified from the soluble fraction by Ni-nitrilotriacetic acidagarose (Qiagen, Valencia, CA) chromatography according to the manufacturer's instructions. For preparation of anti-guinea pig p40 antibodies, 50 µg of purified p40 polypeptide was injected with complete Freund's adjuvant (Difco) into a rabbit every week. Three days after the fourth immunization, antiserum was collected and the polyclonal antibodies were purified by precipitation with ammonium sulfate at 33% saturation.
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Detection of recombinant hybrid IL-12 p70 by ELISA
The levels of various forms of hybrid IL-12 p70 (p35/p40) secreted into the COS cell culture supernatants were determined by ELISA using human IL-12 p70 immunoassay and human IL-12 p40 immunoassay (Genzyme/Techne, Minneapolis, MN) according to the manufacturer's instructions.
Western blotting analysis
For detection of various forms of hybrid p70 secreted into the COS cell culture supernatants or endogenous IL-12 in macrophage culture supernatant, aliquots of 250 µl of culture supernatants were concentrated using an equal volume of 10% trichloroacetic acid and solubilized in reducing or non-reducing sample buffer. Samples were resolved by SDSPAGE and transferred onto PVDF membranes (Millipore, Bedford, MA). The blots were blocked with 5% non-fat milk and treated with rabbit anti-guinea pig p40 polyclonal antibody. After washing, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (BioRad, Hercules, CA) and developed with ECL (Amersham Pharmacia Biotech). In some experiments, the blotted membranes were reprobed by washing twice with 50 mM Tris buffer, pH 6.8, containing 2% SDS and 100 mM 2-mercaptoethanol for 15 min at 50°C, blocked and treated with goat anti-human IL-12 p70 polyclonal antibody (Genzyme/Techne). After washing, the blots were incubated with HRP-conjugated swine anti-goat IgG (Biosource, Lewisville, TX). For detection of endogenous p40 in testis, the tissue was homogenized in 20 mM Tris-buffered saline, pH 7.4, containing 1 mM PMSF, 10 mM EDTA and 1 mg/ml iodoacetic acid. The homogenates were centrifuged and 30 µg of proteins of supernatants were mixed with reducing or non-reducing sample buffer.
IL-12 proliferation assay
Human peripheral blood mononuclear cells (PBMC) were collected with methylcellulose sedimentation followed by centrifugation on a Ficoll-Hypaque cushion. Human PBMC or guinea pig splenocytes (2x106 cells/ml) were cultured in RPMI 1640 containing 10% FCS and 5 µg/ml Con A. After 3 days, the Con A-primed lymphoblasts were harvested, washed and resuspended in RPMI 1640 with 10% FCS at 1.0x106 cells/ml. Aliquots of 200 µl of the cell suspension were mixed with 10 µl aliquots of serial dilution of hybrid p70-containing culture supernatants in 96-well plates. Cells were further incubated for 48 h with [3H]thymidine (New England Nuclear, Boston, MA) added at a final concentration of 1 µCi/well for the last 24 h of incubation. In blocking experiments, the human or guinea pig Con A-primed lymphoblasts (2.0x105 cells) were cultured with 10 µl of human p35/p40-transfected COS cell supernatant in the presence of goat anti-human IL-12 p70 polyclonal antibodies (20 µg/ml) (Genzyme/Techne; cat. no. 42219) or polyclonal goat IgG (20 µg/ml) (Sigma; cat. no. I5256). The assay was performed as described above. All samples were assayed in triplicate.
IFN- induction assay
Human peripheral blood lymphocytes (PBL) (CD14) were isolated from PBMC by negative selection using CD14 magnetic beads (Miltenyi Biotec, Bergish Gladbach, Germany). PBL were suspended in RPMI 1640 containing 10% FCS and 10 ng/ml recombinant human IL-2 (R & D Systems, Minneapolis, MN) at 1.0x107 cells/ml. Aliquots of 200 µl of the cell suspension were mixed with 10 µl aliquots of serial dilution of hybrid p70-containing culture supernatants in 96-well plates, and the cultures were incubated for 36 h at 37°C. The levels of IFN- in culture supernatants were determined by ELISA using human IFN-
ELISA system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. All samples were assayed in triplicate. Production of IFN-
by guinea pig splenocytes in response to hybrid p70 was assessed by Northern blotting analysis. Guinea pig splenocytes were suspended in 10% FCS/RPMI 1640 with or without 10 ng/ml recombinant human IL-2 at 4.0x106 cells/ml. Aliquots of 3 ml of the cell suspension were mixed with 30 µl aliquots of hybrid p70-containing culture supernatants (10-fold) in six-well plates. The cultures were incubated for 36 h at 37°C. After incubation, cells were harvested and lysed in Trizol. RNA extraction and Northern blotting analysis were conducted as described above.
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Results |
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The predicted guinea pig IL-12 p35 was a 221-amino-acid precursor protein with a 22-amino-acid signal sequence (as determined using the signal IP program). Human IL-12 p35 has been reported to consist of two protein isoforms (p35 short and p35 long) yielded by alternative usage of the translational initiation Met codon (20,21). Guinea pig p35 was a homologue of human p35 short, since the initiation Met codon at the appropriate position for p35 long was absent in guinea pig p35 (data not shown). Recognition of a monobasic cleavage site for disparate processing of the signal peptide of IL-12 has been reported in human p35 (22). This site, His18, of human p35 short was not conserved in the guinea pig p35. The precursor form of p35 possessed nine Cys residues and four possible N-glycosylation sites. The primary sequence of guinea pig p35 was compared with those of human, mouse and other species (Fig. 2a). Seven of the nine Cys residues and one of the four N-glycosylation motifs were conserved among the all species examined. Incidentally, the complete HXXLAR (X = any residue) sequence in the putative monobasic cleavage site described above was only conserved among human, mouse, horse and cat p35 subunits. The homology (%) toward guinea pig p35 subunit is 5474% among the species examined and the low score tendency was observed in the rodent (mouse and rat) counterparts. On the contrary, the highest score was observed in human p35.
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Expression of IL-12 subunit mRNA in guinea pig tissues
Northern blotting analysis using the guinea pig p40 and p35 ORF cDNAs as probes was performed to assess the signal intensity in each organ, and in mitogen-stimulated macrophages and splenocytes of guinea pigs (Fig. 3a). Predominant p35 mRNA (2.0 kb) expression was found in the testis, and p40 mRNA in the testis (2.5 kb) and to lesser levels in the kidney (2.02.2 kb). The transcript size of the p40 message in the testis and TGC-elicited macrophages was slightly bigger than those of kidney and cDNA clones. We analyzed the nucleotide sequences of p35 and p40 expressed in the testis by the 5' and 3' RACE technique to confirm that the ORF of these subunits (p35; 168666 bp, p40; 49999 bp, Fig. 1
) were identical to those obtained from macrophages. No significant expression of IL-12 message was found in the lymphoid tissues (thymus and spleen). Scarozza et al. reported that p40 mRNA levels were low or undetectable in intact splenocytes, but increased as a function of time after stimulation with Con A (23). Under our experimental conditions, however, Con A-stimulated spleen cells did not express IL-12. Likewise, no significant difference of IL-12 (both p35 and p40) message was observed between unstimulated and LPS-stimulated macrophages.
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Characterization of recombinant hybrid IL-12 p70
For the following IL-12 bioassays, COS7 cells were co-transfected with vectors expressing each subunit of human or guinea pig IL-12 (hp35/hp40, gp35/gp40, hp35/gp40 and gp35/hp40), and the amounts of recombinant hybrid p35/p40 in the culture supernatants were assessed by ELISA for human p70 and p40 (Fig. 4a). Incidentally, essential amino acids for heterodimerization in the two subunits are all conserved across humans, guinea pigs and mice based on recent crystal structure analysis (27). Only supernatants from COS cells co-transfected with hp35 and hp40 (hp35/hp40) or gp35 and hp40 (gp35/hp40) resulted in detection of heterodimer p70 (18.5 versus 0.4 ng/ml) as well as p40 monomer or homodimer (1.5 versus 3.0 ng/ml). No other combination of transfection including mock transfection allowed us to detect the production of p70 or p40 in these human ELISA systems.
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IL-12-dependent lymphoblast proliferation assay
The growth response of human PBMC and guinea pig spleen cells to various forms of IL-12 p70 hybrid was examined by cell proliferation assay (Fig. 5a and b). [3H]Thymidine incorporation was measured after treatment of lymphocytes with Con A and then the COS cell culture supernatants containing recombinant p70 hybrids. All hybrids induced both human and guinea pig Con A-blast proliferation in a dose-dependent manner. In guinea pig Con A blasts, the growth rates induced by hp35/hp40 and gp35/hp40 were indistinguishable, and slightly higher than those by hp35/gp40 and gp35/gp40. In addition, the neutralizing anti-human p70 antibodies completely blocked the proliferation of both human and guinea pig-hp35/hp40-treated lymphoblasts (Fig. 5c
). Taken together, these growth responses were clearly dependent on recombinant p35/p40.
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IL-12-dependent IFN- induction assay
IFN- levels in the culture supernatants of human PBL treated with various p70 hybrids plus human rIL-2 were measured by ELISA (Fig. 6a
). The levels of IFN-
induced by the hybrids were very high in hp35/hp40, moderate in gp35/hp40 and hp35/gp40, and low but significant in gp35/gp40. A similar tendency was observed solely with the hybrid (no addition of IL-2) although the efficiency of IFN-
production was very low (Fig. 6b
). Taking these results together, all of the p70 hybrids induced IFN-
production by human PBL and synergized effectively with IL-2.
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Discussion |
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IL-12 is a major innate immune factor produced predominantly by macrophages and other APC, and functionally encompassing innate and acquired immunity for balancing of optimal cell-mediated immune responses (1,2). It has been reported that the p35 subunit governs the species specificity of IL-12 function (4,5). This concept was proposed based on the results with hybrid heterodimers (p70) consisting of human and mouse p35 and p40 subunits. However, this concept is not the case between human and non-human primates, and based on the present results cannot be generalized between humans and rodents (6,7). In this study, functional complementation was established with hybrid heterodimers consisting of human and guinea pig p35 and p40 subunits. Both human PBMC (or PBL) and guinea pig splenocytes responded to p70 hybrids with different sensitivity in terms of cell proliferation (Fig. 5) and IFN-
production (Figs 6 and 7
). Accordingly, guinea pig IL-12 must be a functional substitute for human IL-12. This is in part attributable to the high degree of structural similarity between human and guinea pig IL-12 subunits. As COS7 cells expressed not only p70 but also free p40 (monomer or homodimer) (Fig. 4ac
), the differences in sensitivity of hybrid p70 between human PBMC (or PBL) and guinea pig splenocytes may be due to differences in binding affinity of antagonistic p40 homodimers to the receptor (1113). Therefore, the molecular structure of the IL-12 receptor and intermolecular association between IL-12 and its receptor complex must be elucidated in guinea pigs.
The tissue distribution profile of the guinea pig IL-12 based on the results of both northern and Western blotting analyses is intriguing (Fig. 3). There have been no previous reports that the testes constitutively express p35 and p40 messages and the p40 protein. It is notable in this study that the p40 protein was the main product and the unidentified 200-kDa macromolecule antigenically related to the p40 protein was found in the testis. Judging from the analogy of IL-12 to IL-6 family proteins, the relationship between p35 and p40 should be a ligandreceptor interaction. Thus, it is not surprising that each subunit of IL-12 can couple with different partners. Actually, p35 forms a complex with an EpsteinBarr virus-induced gene (EBI3), i.e. p35/EBI3, whose function has not yet been identified (30). p40 can choose a p19 (19-kDa) protein, closely related to p35, as a disulfide-bridged partner to exert IL-12-like cytokine activity(31). This p19/p40 complex was named IL-23 and its functional profile was found to be in part distinct from that of IL-12. Hence, the relevant 200-kDa molecule may reflect the presence of an alternative candidate of the p40-containing proteins. Analysis of the composition of the 200-kDa molecule is in progress in our laboratory.
The role of IL-12 in the testis also remains to be clarified. A variety of cytokines have been reported to have roles as paracrine mediators under physiological or pathophysiological conditions in the testis of rodents. IL-1 is constitutively expressed in rat Sertoli cells and its expression levels are dependent on the stage of the seminiferous epithelial cycle; both the IL-1 bioactivity and message were diminished in stage VII of the cycle or in the seminiferous tubules depleted of germ cells (32). Accordingly, some reports suggested that IL-1
may participate in DNA replication in the testicular germ cells (3234). Regarding IL-12, only one preliminary report suggested that rat Leydig cells cultured in vitro produce the IL-12 p40 subunit in a luteinizing hormone (LH) concentration-dependent manner in parallel with testosterone induction (35). The testosterone induced by LH is considered to act on the stage VII Sertoli cells, which in turn participate in paracrine regulation of germ cells. However, the function of this inducible IL-12 in the testis and distribution of the IL-12 receptor in the testis remain unknown, and therefore further analyses are needed to evaluate their physiological significance in the testis.
Mice have been used as an animal model for human immune diseases as their gene disruption procedure was established. However, as a human model, mice are somewhat different, particularly in the innate immune system. The complement system differs between mice and humans (36). Toll-like receptor (TLR) 2 activation leads to killing of intracellular Mycobacterium tuberculosis, but the mode of bactericidal activity again differs between murine and human macrophages (37). Species differences are also observed in APC-derived initial cytokines including IL-23 (31). At least, these innate factors are difficult to analyze using the mouse as a human analogue. In contrast, many studies of the comparative biology of the guinea pig have revealed a number of marked similarities between this species and humans, especially in the field of innate immunity (14). These fundamental similarities bear directly or indirectly on the relevance of the guinea pig as a species to model human infectious disease, especially TB (1416). It has been accepted that the guinea pig, as well as the mouse and rat, belongs to the rodent family. However, recent molecular phylogenic studies suggested that the guinea pig may not be part of the order Rodentia (38,39) and also support this concept regarding the IL-12 molecule in this study (Fig. 2 and phylogenic analyses, data not shown). In addition, molecular cloning of guinea pig CD1 suggested that guinea pigs have eight isoforms which are homologues of human group 1 CD1b, CD1c subclasses and CD1e, while muroid rodents (mice and rats) have only homologues of human group2 CD1d subclasses (40). CD1 is a family of non-polymorphic genes that seems to have evolved to present lipid and glycolipid antigen including lipoarabinomannan and mycolic acids of TB (41). As group 1 and group 2 CD1 proteins have distinct roles in the host immune responses with respect to the types of antigens presented by each group, this issue may provide a key to the analysis of the different responses of guinea pigs and mice to TB (14,15).
A regrettable point is that compared to the mouse or human immune system, the guinea pig immune system lags far behind in its analysis. Molecular cloning of guinea pig cytokines, surface adhesion molecules and innate immune-related molecules should help establish guinea pig as a model animal. We have cloned IL-18 and IL-23 (the p19 subunit) as well as IL-12. Myd88, an adaptor molecule for TLR, was also cloned (Shiratori et al., unpublished). Although these results are not published in this communication, accumulating evidence supports our idea that the guinea pig innate system more resembles that of humans than that of mice.
In summary, IL-12 is involved in the initial steps of infection, allergy and protection against tumor progression. Most studies including gene therapy are being developed using mice. Here, we propose that the guinea pig will be an alternative small animal model for clinical application of IL-12 and will provide certain advantages over the mouse in some points, especially in vivo studies analyzing the effects of human rIL-12 in infectious disease, allergy and cancer. Further molecular analyses will be needed to confirm this proposal.
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Acknowledgments |
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Abbreviations |
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Con A concanavalin A |
CTL cytotoxic T lymphocyte |
G-CSF granulocyte colony-stimulating factor |
HRP horseradish peroxidase |
LH luteinizing hormone |
LPS lipopolysaccharide |
ORF open reading frame |
PBL peripheral blood lymphocyte |
PBMC peripheral blood mononuclear cell |
R receptor |
RACE rapid amplification of cDNA ends |
TB tuberculosis |
TGC thioglycollate medium |
TLR Toll-like receptor |
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Notes |
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Received 2 April 2001, accepted 30 May 2001.
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References |
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