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GeneMedicine, Inc., The Woodlands, TX 77381
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Abstract |
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 Top Abstract IntroductionMaterials and Methods ResultsDiscussionReferences |
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and
IL-12 in bronchoalveolar lavage fluid is caused by unmethylated CpG
dinucleotide sequences present within the plasmid, and is enhanced by
the lipid formulation; 2) cationic lipids by themselves do not induce
IL-12 or IL-12p40; 3) TNF-
is rapidly induced by cationic lipids and
plasmid/lipid complex, but not by plasmid alone; 4) an acute cellular
influx is induced by cationic lipid alone and by a plasmid/lipid
complex, but to a much lesser extent by plasmid alone; and 5) plasmid
methylation does not influence the degree of inflammatory cell influx.
The induction of the innate immune responses by plasmid/lipid complexes
may be advantageous to gene therapy of lung diseases. In particular,
induction of the Th1 cell-promoting cytokines by plasmid/lipid
complexes could, in conjunction with an expressed transgene, be used to
modulate immune responses in the lung airways in disease conditions
that are deficient in Th1 cell responses or that have a dominant Th2
phenotype. Alternatively, the elimination of immunostimulatory
sequences in plasmids may improve the tolerability and/or efficacy of
nonviral gene therapy, especially for diseases requiring chronic
administration. |
Introduction |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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1-antitrypsin deficiency is currently being tested
in human clinical trials (1, 2, 3). While the expression in vivo of genes
transfected in human and animal lung tissue using cationic lipids has
been well documented (4, 5, 6), recent clinical trials also showed that
the administration of plasmid formulations induce other effects (e.g.,
inflammation) that must be considered (7). There is clearly a need for
elucidating various mechanisms associated with the effects of plasmid
formulations before safe and effective gene medicines can be
developed.
It is known that in vitro and in vivo cellular immune responses to
bacterial DNA (8, 9) and plasmid (10, 11, 12) are associated with specific
CpG dinucleotide sequences. It has been demonstrated that these immune
responses are primarily due to unmethylated CpG dinucleotide motifs
contained within the RRCGYY sequence (13). Bacterial DNA contains about
four to five times as many CpGs as mammalian DNA (14). CpGs are usually
unmethylated in bacterial DNA, whereas in mammalian DNA some 75% of
the CpGs are methylated to 5-methylcytosine (15). Methylation
inactivates CpGs with respect to their immunostimulatory effects (14).
The ratio of unmethylated CpGs contained within the RRCGYY hexamer
sequences in the bacterial and mammalian DNAs is about 20 (14, 16).
This structural difference between bacterial and mammalian DNA is a
signal for the induction of innate immunity to microbial infections
through the induction of Abs (14); generation of a variety of
cytokines, including IFN-, TNF-, and IL-12, which promote
Th1-dependent cell responses; and enhancement of NK cell activity
(17).
Responses to cationic lipids that are frequently used for formulating plasmids and to the plasmid/lipid complexes themselves are much less well understood. In this study, we have examined the contribution of individual components of a plasmid/lipid complex to the immune responses in the lung airway of mice following intratracheal administration. We used N-[1-(2–3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA2)/cholesterol/plasmid 3:1 positively charged complexes that have been shown to enhance pulmonary transgene expression when delivered by intratracheal instillation (D. Deshpande, P. Blezinger, R. Pillai, G. Padmabandhu, J. Duguid, B. Freimark, J. Slater, M. Bruno, K. Petrak, and A. Rolland, manuscript in preparation). The identification of the components of plasmid/cationic lipid complexes that are responsible for inducing specific immune responses is important for the development of safe and effective nonviral gene therapies for the treatment of pulmonary disorders, and is the subject of this report.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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pCT0129 is an expression plasmid derived from a vector containing the human CMV immediate-early promoter (18) (Clontech, Palo Alto, CA) with the ß-galactosidase gene removed and a NotI-linked chloramphenicol acetyltransferase (CAT) gene inserted in its place. pVC0289 is a cloning vector with no cDNA insert based on pBluescript KS+ (Stratagene, San Diego, CA) that replaces the ampicillin resistance gene with the kanamycin resistance gene from pNEOßgal (Stratagene). Specific regions of plasmid were isolated by digestion with restriction endonucleases (New England Biolabs, Beverly, MA; and Promega, Madison, WI) and purified by HPLC as described below. A 1008-bp fragment containing the ß-lactamase promoter and ampicillin resistance gene was isolated from pBluescript KS+ using BspHI. A 1149-bp fragment containing the Tn5 promotor and kanamycin resistance gene was isolated from pVC0289 using NotI and DdeI. Two fragments, 673 bp and 409 bp, containing the pUC origin of replication were isolated from pBluescript KS+ using Acc65I and DdeI. A 548-bp fragment containing the CMV enhancer/promoter was isolated from pCMVß using EcoRI and HindIII. A 302-bp fragment encoding the amino-terminal portion of human IL-2 cDNA lacking CpG sequences was isolated from an IL-2 expression plasmid using BamHI and AflII. Plasmid (1–2 mg) was digested with a 10-fold excess of restriction endonuclease and digestion was confirmed by agarose gel electrophoresis before purification. Calf thymus DNA (Sigma, St. Louis, MO) was digested with EcoRI before HPLC purification. Escherichia coli genomic DNA (Sigma) was used without further purification. Analysis of CpG frequency in plasmid and plasmid fragments was determined using the VectorNTI software package (Informax, Gaithersburg, MD).
Plasmid methylation
pCT0129 (1–2 mg) was methylated with SssI CpG methylase (New England Biolabs) using 5 U of enzyme/µg DNA for 4 h and incubated for an additional 2 h following the further addition of S-adenosylmethionine substrate using buffer conditions suggested by the manufacturer. DNA was extracted with phenol-chloroform and precipitated with ethanol after methylation. The efficiency of methylation was confirmed to be >95% by enzyme digestion using BstUI, HhaI, HpaII, and MspI. DNA was further purified by HPLC chromatography.
DNA purification
Intact supercoiled plasmid was extracted from bacterial lysates by alkaline lysis and isolated by conventional chromatography techniques. Plasmid fragments, methylated plasmid, and restriction endonuclease-digested E. coli or calf thymus genomic DNA (Sigma) were purified by HPLC chromatography using a Hewlett Packard 1050 series HPLC unit (Hewlett Packard, Palo Alto, CA) interfaced with a BioCADJ Workstation (PerSeptive Biosystems, Cambridge, MA). Restriction endonuclease-digested plasmids (50–100 µg) were applied to a TSKgel DNA-NPR column (ToSoHaas, Montgomeryville, PA) and eluted using a 20-min salt gradient generated from 0.5 to 0.75 M NaCl containing 10 mM Tris-HCl (pH 8) and 1 mM EDTA at a flow rate of 0.5 ml/min. The EcoRI-digested calf thymus and E. coli genomic DNAs (1 mg) were purified using a 1-ml RESOURCE Q column (Pharmacia Biotech, Piscataway, NJ). DNA was applied to the column and then eluted using a 10-bed volume salt gradient formed from 0.5 M NaCl to 0.85 M NaCl containing 10 mM bis-Tris propane buffer (pH 7.5) at a flow rate of 8 ml/min. Purified DNAs were concentrated by ethanol precipitation. The purity of plasmid fragment isolation was assessed to be greater than 95% by agarose gel electrophoresis. Endotoxin levels were determined using a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). Levels of endotoxin were less than 50 EU/mg DNA. In some studies, endotoxin (E. coli subtype 055:B5; Sigma) was spiked into DNA before complexation with cationic liposomes.
Preparation of liposomes and DNA/lipid complexes
Small unilamellar vesicles, composed of the cationic lipid
DOTMA:cholesterol at a 1:1 M ratio (hereafter referred to as lipid),
were prepared by extrusion. Positively charged plasmid/lipid complexes
were prepared at a 1:3 -/+ charge ratio in 10% (w/v) lactose by
mixing the plasmid, plasmid fragment, or calf thymus DNA with the
liposomes under controlled conditions (D. Deshpande, P. Blezinger, R.
Pillai, G. Padmabandhu, J. Duguid, B. Freimark, J. Slater, M. Bruno, K.
Petrak, and A. Rolland, manuscript in preparation; and Refs. 19
and 20). The DNA concentration in the formulation was 100 µg/ml. The
mean diameter and 
potential of the complexes were characterized
using dynamic light scattering and Doppler electrophoretic light
scattering. The complexation efficiency was determined by agarose gel
electrophoresis (D. Deshpande, P. Blezinger, R. Pillai, G. Padmabandhu,
J. Duguid, B. Freimark, J. Slater, M. Bruno, K. Petrak, and A. Rolland,
manuscript in preparation).
Animals
Male C57BL/6 mice (20–25 g; Harlan Laboratories, Houston, TX) were maintained on ad libitum rodent feed and water at 23°C, 40% humidity, and a 12-h/12-h light-dark cycle. Animals were acclimated for at least 3 days before the start of the study.
In vivo administration
Animals were anesthetized with 30 µl of ketamine (Fort Dodge Animal Health, Overland Park, KS; 150 mg/kg) i.p. They were suspended in a vertical position and received either a 50-µl single bolus of formulation in 10% lactose or 10% lactose alone in the trachea using a 24-gauge gavage cannula. This volume of formulation has been found to produce the optimal gene expression with minimum lung trauma or animal mortality (data not shown).
Tissue harvest and extraction
Bronchoalveolar lavage fluid (BALF) and/or lung tissue were collected at various time points following administration. Animals were euthanized with a 50-µl mixture containing 73.96 mg/ml ketamine, 3.74 mg/ml xylazine, and 0.73 mg/ml acepromazine. For BALF collection, the trachea was exposed by making a mid-line incision, then cannulated endotracheally with a 22-gauge gavage needle (VWR, Houston, TX) attached to a 1-ml syringe, and lavaged twice with 1-ml aliquots of HBSS (Life Technologies, Grand Island, NY) without Ca2+ or Mg2+. Washes were pooled, centrifuged for 1 min at 200 x g, and the cellfree supernatant stored at -80°C. For determining cell influx counts, RBCs were removed by hypotonic shock using 0.1 ml of distilled water followed by the addition of 0.9 ml of HBSS. For preparation of total lung extracts, excised lung lobes were transferred to 2-ml screw-cap polypropylene tubes containing 0.25 g of zirconium beads (Biospec, Bartlesville, OK) and snap-frozen in liquid nitrogen for storage at -80°C. Protein was extracted from tissue by bead homogenization (Biospec) with 1 ml of ice-cold extraction buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 µM pepstatin A, 0.25 mM PMSF, and 10 µM leupeptin). The tubes were centrifuged for 10 min at 4°C before immunoassay. Blood was obtained from animals by cardiac puncture and the serum was separated using serum separation tubes (catalog no. 5960; Becton Dickinson, Franklin Lakes, NJ).
In vitro cell culture
Tissues harvested from animals that were administered various
formulations were cultured at 2 x 106/ml in DMEM
supplemented with 10% FCS, sodium pyruvate, and antibiotics for
48 h at 37°C. Splenocytes were prepared by 0.83%
NH4Cl lysis of RBCs (19). Lung airway cells were prepared
by digestion with bovine pancreatic protease (Sigma) and DNase I
(Sigma) (20). In some cultures, recombinant mouse IL-12, IL-2, and
TNF- (R & D, Minneapolis, MN) were added to activate cells. Culture
supernatants were harvested and assayed for the presence of various
cytokines by immunoassay.
Immunoassays
Cytokine levels in BALF and lung tissues were determined using
specific immunoassay kits for mouse IL-12p40 and mouse IL-12p70
(Genzyme, Cambridge, MA), mouse IFN-, mouse IL-1ß, mouse IL-4, and
mouse TNF- (Endogen, Woburn, MA). The sensitivity of these assays
was 10 to 15 pg of cytokine/ml. The IL-12p40 immunoassay detects the
total IL-12p40 that is present as a monomer, homodimer, or heterodimer,
whereas the IL-12p70 immunoassay detects only the heterodimer.
Expression levels of CAT were determined in lung extracts using a
specific immunoassay (Boehringer Mannheim, Indianapolis, IN). Samples
were analyzed in duplicate on a plate reader (model EL340; Bio-Tek
Instruments, Winooski, VT) and cytokine levels were calculated by
linear regression analysis (KC3; Bio-Tek Instruments) based on values
obtained from a standard curve. The intra- and interassay coefficient
of variation of sample readings was <10%.
Statistical analysis
Data were analyzed by the Mann-Whitney test using the SPSS Base 7.5 for Windows statistics software package (SPSS, Chicago, IL). Data were considered statistically significant if p values were < 0.05.
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Results |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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A summary of the number and frequency of CpG dinucleotide motifs
present in plasmid and genomic DNA used in our studies is shown in
Table I
. Plasmids replicated in E.
coli host cells typically contain CpGs at similar frequencies (1
per 16 dinucleotides) to bacterial DNA. In contrast, the CpGs are only
about one-quarter as prevalent in mammalian DNA (14).
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, intact plasmid and various fragments of plasmids were
formulated with lipid and administered by intratracheal instillation.
Total IL-12 (p40 monomer, homodimer, or heterodimer) and IFN-
production was assessed in the lung 24 h following administration
of DNA/lipid complexes with a 1:3 (-/+) complex charge ratio.
Increased levels of endogenous IL-12 and IFN- were observed in lung
tissues from animals administered plasmid/lipid complexes using two
different plasmids (Fig. 1A) compared with a
control group receiving 10% lactose (Fig. 1A). We
obtained similar results with the same lipid formulation using other
plasmids (data not shown). Administration of restriction
endonuclease-digested plasmids formulated with cationic lipid did not
reduce the levels of cytokine (Fig. 1B). Instillation
of purified plasmid fragments consisting of the replication of origin,
CMV promoter/enhancer elements, kanamycin resistance gene, ampicillin
resistance gene, and bacterial genomic DNA (Fig. 1B)
all resulted in the induction of IL-12. In contrast, lung tissue from
animals instilled with calf thymus DNA/lipid complexes had low levels
of IL-12. Animals receiving lipid alone had IL-12 levels that were
indistinguishable from untreated controls.
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The induction of IL-12 and IFN- by CpG motifs was further
evaluated by comparing the responses to mammalian DNA, methylated
plasmid, or a plasmid fragment lacking CpG motifs. Intratracheal
instillation of complexes consisting of lipid and mammalian DNA or
methylated plasmid or a CpG-deficient plasmid fragment from the human
IL-2 gene induce about 10-fold less IL-12 and IFN- compared with
animals treated with unmethylated plasmid/lipid complexes (Fig. 2). These observations are consistent
with the previous studies, which show that the key element in induction
of IL-12 and IFN- is the presence of unmethylated CpG motifs in
plasmids (8, 9, 10, 11).
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The contribution of separate plasmid and lipid components to the
induction of cytokines was measured. For these studies, a 5-µg dose
of plasmid was administered, since IL-12p40 and IFN- were clearly
induced when administered to the lungs as a plasmid/lipid complex
(Figs. 1
and 2). We show that administration of plasmid/lipid complexes
induces IL-12p40, IL-12p70, TNF-, and IFN- (Fig. 3A) compared with
animals instilled with isotonic 10% lactose (Fig. 3A). Levels of total IL-12 and IFN- were higher at
24 h than 2 h following administration. Peak levels of
TNF- induced by either lipid alone or plasmid/lipid complexes were
observed at 2 h and decreased by 24 h following
administration. Instillation of lipid in 10% lactose induced low
levels of IL-12p70, but IL-12p40 or IFN- was not detected (Fig. 3A). Administration of plasmid in 10% lactose
induced low levels of IL-12p40 and IL-12 p70, but not TNF- or
IFN- (Fig. 3A). The levels of IL-1ß and IL-4
were not enhanced in BALF of animals administered plasmid/lipid
complexes compared with controls (data not shown). The data show that
the lipid alone does not induce IL-12 and IFN- but induces TNF-.
Plasmids alone induce IL-12 (both p40 and p70) but not IFN- and
TNF-.
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Cellular influx was determined 24 h following the
administration of the separate plasmid and lipid components and the
plasmid/lipid complexes. The highest cell influxes in the airway were
seen in animals given the lipid alone or plasmid/lipid complexes (Fig. 3B). Over 90% of the cells recovered from lungs were
neutrophils and macrophages. No differences in the cell influx were
observed when methylated or unmethylated plasmids complexed with lipid
were compared with the lipid alone (data not shown). We conclude that
the lipid component of the plasmid/lipid formulation is primarily
responsible for the induction of cell influx into the airway.
Production of cytokines by resident and infiltrating cells of the lung
To ascertain whether cytokines induced in the lungs were secreted
by cells in the lung airways or from a distal site such as the
circulation, BALF cells from animals administered with plasmid/lipid
complexes were cultured in vitro and the supernatants assayed for
cytokines. BALF cells harvested from animals administered plasmid/lipid
complexes or plasmid alone secreted IL-12p40, TNF-, and IFN- in
vitro (Fig. 4), but not IL-12p70. TNF-
and IFN- were highest in culture supernatants from animals
administered plasmid/lipid complexes, whereas IL-12p40 levels were
similar among all the plasmid treatment groups. The serum level of
IFN- among all plasmid treatment groups was similar (Fig. 5). Since IFN- in the BALF is diluted
approximately 20-fold (
100 µl diluted to 2 ml), the most likely
source of this cytokine is in situ production. In the same group of
animals, expression of CAT transgene was measured in the lung tissue as
an indirect measurement of plasmid internalization (Fig. 6). Animals receiving 50 µg of plasmid
alone had similar expression levels to animals receiving 5 µg of
plasmid formulated with lipid. Administration of lower doses of
plasmids alone or plasmid formulated with lipid resulted in
significantly lower expression levels. If the level of expression is
directly proportional to the copy number of plasmids internalized, then
our data suggest that cytokine induction is not entirely dependent on
internalization of plasmid. To determine whether the cytokines induced
by plasmid/lipid complexes costimulate cells to produce IFN-,
splenocytes and crude epithelial lining cell preparations were
stimulated with TNF-, IL-12, or both together (Fig. 7). Stimulation of splenocytes with
recombinant mouse IL-12 induced low levels of IFN- and was enhanced
by the presence recombinant mouse IL-2 or TNF-. TNF- did not
induce detectable levels of IFN- in splenocyte cultures. Stimulation
of crude lung airway epithelial cells with IL-12 alone secreted IFN-
into the culture supernatant. Compared with splenocytes, there did not
appear to be a costimulatory effect of TNF- to IL-12-dependent
IFN- secretion.
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Instillation of endotoxins is known to induce proinflammatory
responses in the lung, including induction of IL-12, TNF-, and
IFN- (21, 22). Thus, contamination of plasmid/lipid complexes by
endotoxin could contribute to the induction of cytokines observed in
this study. To test this, plasmid and calf thymus DNA were spiked with
increasing amounts of purified E. coli endotoxin before
complexing with lipid. The original level of endotoxin was 0.002 EU per
5 µg of plasmid and 0.129 EU per 5 µg of calf thymus DNA. Plasmid
and calf thymus DNA were spiked with 1, 10, or 100 EU of endotoxin
before complexing with lipid, and were then administered to animals.
Cell influx and cytokine levels in BALF were determined at 24 h
following administration. No increase in IL-12 and IFN- levels over
the levels induced by plasmid/lipid complexes was seen even at the
highest (i.e., 100 EU) dose of endotoxin (Fig. 8A). IFN- and
IL-12p40 were found in BALF only when bacterial endotoxin was added to
the calf thymus DNA formulation at a level that was 500-fold higher
than the amount detected in purified plasmid. Significant increases in
BALF cell counts were observed in animals receiving at least 1 EU of
endotoxin added to plasmid or 10 EU of endotoxin added to calf thymus
DNA complexed with lipid (Fig. 8B), i.e., at levels
of endotoxin that were much higher than initially present in the
plasmid and calf thymus DNA.
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Discussion |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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A).
For the same dose of plasmid (5 µg), IFN- and TNF- were not
detected in lavage fluids. IFN- was detectable only when larger
doses (50 µg) of plasmid were administered. In other studies,
instillation of 300 µg of plasmid alone in rat lungs induced
detectable levels of TNF- in the BALF (24). It is further shown that
exposure of C3H mice to 10 µg of bacterial genomic DNA or
immunostimulatory oligonucleotides can stimulate TNF-, IL-6,
macrophage inflammatory protein-2, and increased BALF cell counts (25).
Taken together, it appears that a threshold dose of 1 to 5 µg of
plasmid is required to elicit immunostimulatory responses in the lung.
Data obtained from the these studies have shown that the immune
response to plasmid or plasmid/lipid complexes used in this study is
not due to bacterial endotoxin at levels present in plasmid
preparations.
The immune responses elicited by plasmid were markedly
enhanced when administered as a positively charged complex
with cationic lipids. Plasmid/lipid complexes induce Th1-promoting
cytokines, such as IL-12 and IFN-. The induction is dependent on the
presence of unmethylated CpG motifs. We have shown that administration
of a plasmid fragment lacking CpG motifs, complexed with the lipid,
does not induce IL-12 and IFN-. It is likely that the stimulation of
macrophages and neutrophils by plasmid/lipid complexes leads to IL-12
production, since both cell types have been demonstrated to produce
this cytokine (26, 27, 28, 29, 30). By histologic analysis, low numbers of
lymphocytes were observed in the proinflammatory cell influxes. It is
not clear if these cells or other cell types, such as respiratory
epithelial cells, are responsible for IFN- production (31). Our data
indicate that inflammatory cells infiltrating the lung following the
administration of plasmid/lipid complexes consisted primarily of
macrophages and neutrophils. In vitro culture of BALF cells
demonstrates that these cells are capable of continued synthesis of
TNF-, IL-12p40, and IFN- without further stimulation. In
addition, in vitro stimulation of crude epithelial lining cells from
untreated mice with IL-12 induces IFN- production. These data
suggest that the likely source of cytokines observed in the BALF were
the resident and infiltrating inflammatory cells.
The enhanced immune responses by plasmid/lipid complexes may be due to
several mechanisms. First, the immune responses to the separate plasmid
and cationic lipid components produce two different sets of cytokines.
Several studies have shown that IL-12, which is produced mainly by
cells of the monocyte/macrophage lineage (28), is instrumental in the
production of IFN- by activated T cells and NK cells (32, 33). In
addition, TNF- has been to shown to enhance IL-12-dependent IFN-
production in vitro (34, 35) and in disease states (36, 37, 38). Hence, the
induction of IFN- by plasmid/lipid complexes and not by the
individual components may be the combined result of induction of
costimulatory cytokines such as TNF- and IL-12. Data obtained in
this study suggest that IL-12 alone is capable of stimulating IFN-
production in normal lung epithelial cell preparations and has no
costimulatory effect of TNF-. In contrast, stimulation of
splenocytes with IL-12 and TNF- or IL-2 resulted in higher IFN-
production than IL-12 alone. It is not known whether this represents an
in vitro artifact or whether IFN--producing cells of the lung
require different stimulatory signals. Second, plasmid/lipid complexes
may enhance the induction of immune responses through increasing
intracellular uptake. This view is supported by the observation that
oligonucleotides immobilized on latex beads, which could not be taken
up by cells, did not lead to cell activation (14). In addition, the
cell uptake of stimulatory oligonucleotides when complexed with
Lipofectin has been shown to induce IFN- and NK activity 3000-fold
more efficiently than plasmid alone (13). Our data show that the level
of expression of a transgene in the lung (Fig. 6) correlates with
elevated BALF cytokine levels (Fig. 5). Third, it is known (4) that
plasmids degrade more slowly in the lung airways when complexed with
lipids. The resulting increased persistence of plasmid in the airways
could enhance both of the above mechanisms. Taken together, the
induction of immune responses by plasmid/lipid complexes bears a
striking resemblance to the innate immunity induced by bacterial
infections in the lung (21, 22, 39, 40, 41, 42, 43) and is likely a composite
response of intra- and extracellular triggering events.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DOTMA, N-[1-(2–3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; BALF, bronchoalveolar lavage fluid, CAT, chloramphenicol acetyltransferase.
Received for publication August 15, 1997. Accepted for publication January 7, 1998.
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References |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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production in murine splenocytes: role of natural killer cells and macrophages. J. Immunol. 150:3973.[Abstract]
and TNF-. J. Immunol. 157:798.[Abstract]
synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533.[Abstract]
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 D. C. Siess, C. T. Vedder, L. S. Merkens, T. Tanaka, A. C. Freed, S. L. McCoy, M. C. Heinrich, M. E. Deffebach, R. M. Bennett, and S. H. Hefeneider A Human Gene Coding for a Membrane-associated Nucleic Acid-binding Protein J. Biol. Chem., October 20, 2000; 275(43): 33655 - 33662. [Abstract] [Full Text] [PDF]
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