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Multiorgan Transplant Program and Departments of
*
Medicine,
Surgery, and
Pathology, The Toronto Hospital and The University of Toronto, Toronto, Canada M5G 2C4
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß all decreased to basal
concentrations: PCA from 941 ± 80 to 34 ± 11
mU/106 cells; TNF- from 10.73 ± 2.15 to
2.74 ± 0.93 ng/ml; and IL-1ß from 155.91 ± 22.62 to
5.74 ± 0.70 pg/ml. The inhibitory effects of ribavirin were at
the level of gene transcription as evidenced by Northern analysis. Both
in vitro and in vivo, ribavirin inhibited the production of IL-4 by Th2
cells, whereas it did not diminish the production of IFN-
in Th1
cells. In contrast, ribavirin had no inhibitory effect on TNF- and
IL-1ß production in LPS-stimulated macrophages. These results suggest
that the beneficial effects of ribavirin are mediated by inhibition of
induction of macrophage proinflammatory cytokines and Th2 cytokines
while preserving Th1 cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous reports have shown that ribavirin attenuates the course of an experimental model of fulminant hepatitis in mice produced by murine hepatitis virus strain 3 (MHV-3)3 (4). Studies in our laboratory as well as others have demonstrated a causal relationship between macrophage activation, production of proinflammatory mediators such as TNF and IL-1 as well as the unique procoagulant activity fgl2 prothrombinase, and the subsequent development and pathogenesis of MHV-3-induced liver disease (5, 6).
To examine the mechanisms of the beneficial effect of ribavirin in this experimental model of viral hepatitis, we studied its effects on MHV-3 replication; its effects on macrophage production of TNF, IL-1, and PCA and Th1/Th2 cytokine production in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/cJ, A/J, and C3H/HeJ mice, 6 to 8 wk of age, were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). The animals were kept in microisolator cages and housed in the animal facilities at the Toronto Hospital, and were fed with standard laboratory chow diet and water ad libitum.
Virus
MHV-3 was obtained from American Type Culture Collection (Rockville, MD) and was plaque purified on monolayers of DBT cells and grown to titers of 2 x 107 plaque-forming units (PFU)/ml in 17 CL1 cells. Virus was harvested by centrifugation at 4500 x g for 1 h at 4°C and was assayed on monolayers of L2 cells in a standard plaque assay (5).
Cells
Peritoneal macrophages were harvested from BALB/cJ mice 4 days after i.p. administration of 1.5 ml of 3% thioglycolate (Difco Laboratories, Detroit, MI) as previously described (6). Macrophages were resuspended in RPMI 1640 (ICN Biomedicals, Costa Mesa, CA) supplemented with 2 mM glutamine (Sigma Chemical, St. Louis, MO) and 2% heat-inactivated FCS (Flow Laboratories, Mississauga, Ontario, Canada). Macrophages were >95% pure as determined by morphology and nonspecific esterase stain (7). Viability exceeded 95% by trypan blue exclusion.
A Th1/Th2 cell line was derived from C3H/HeJ mice that had been immunized with 1 x 108 irradiated (2000 rad) B10BR spleen cells and poly(IC) i.p. T cells were recovered from spleen, diluted, and incubated with 2 x 105 irradiated B10BR cells and IL-2 in 96-well U-bottom plates. Cells were fed weekly by adding fresh medium and rechallenged every 10 days with freshly harvested and irradiated B10BR spleen cells. Wells showing proliferation were transferred to T-25 tissue culture flasks. Stable T cells were then passaged and redistributed at limiting dilution under the same conditions (with fresh medium and irradiated B10BR spleen cells).
CD4+ Th1 (3E9.1) and Th2 (3B6.8) cell lines were derived
from MHV-3-resistant A/J and MHV-3-susceptible BALB/cJ mice,
respectively, as previously described (8). The cell lines were
recovered from draining lymph nodes of mice that had been injected in
the footpad with MHV-3. The cell lines were subtyped by flow cytometry
and cytokine production assayed by the ability of supernatants to
support proliferation of CTLL-2 and CT4.S cells in the presence of
anti-IL-2 and anti-IL-4 mAbs, respectively. IFN- activity
was determined by inhibition of plaque formation induced by MHV-3 on L2
cells (8). Stable T cells were passaged with fresh medium and
irradiated splenic mononuclear cells as described previously
(8).
Viral titer (plaque assay)
The effect of ribavirin on viral titer were determined in a standard plaque assay (5). Briefly, monolayers of peritoneal macrophages from BALB/cJ were pretreated with either ribavirin (0–500 µg/ml) or medium (RPMI 1640), and 2 x 106 PFU of MHV-3 was then added. Following a 10-h incubation at 37°C with 5% CO2, cells were harvested, subjected to one cycle of freeze-thawing, and assayed for viral titers on monolayers of L2 cells.
PCA
MHV-3-infected macrophages, at a multiplicity of infection (MOI) of 2.5, were incubated with ribavirin for 8 h in supplemented RPMI 1640. Uninfected macrophages and MHV-3-infected macrophages without drug treatment were set up as negative and positive controls, respectively. Macrophages were evaluated for functional PCA in a one-stage clotting assay as previously described (5). Following incubation, samples to be assayed for PCA were washed three times with unsupplemented RPMI 1640 and resuspended at a concentration of 106/ml. Samples were assayed for the ability to shorten the spontaneous clotting time of normal citrated human platelet-poor plasma. Milliunits of PCA were assigned by reference to a standard curve generated with serial log dilutions of a standard rabbit brain thromboplastin (Dade Division, American Hospital Supply, Miami, FL). Media and reagents were assayed showing no PCA activity.
Cytokine assay
TNF.
MHV-3 (at a MOI of 2.5) or LPS (10 µg/ml, Sigma)-stimulated BALB/cJ
macrophages were incubated with ribavirin (0–500 µg/ml) for 8 or
4 h, respectively. Supernatants were collected, and TNF-
concentrations were assayed by ELISA. Monoclonal hamster
anti-murine TNF- Ab (Genzyme, Boston, MA) was coated to ELISA
plates overnight at 4°C. After being washed with Tris buffer
(pH 8.0), plates were blocked with 100 µl of 6% BSA in each
well for 1 h at room temperature. Following washing, 100 µl of
standards or samples were added and incubated at room temperature for
3 h. Subsequently, 100 µl of polyclonal rabbit anti-mouse
TNF- Ab (Genzyme, IP-400) were added to each well, and plates were
incubated at 4°C overnight. Goat anti-rabbit IgG alkaline
phosphates (100 µl) (Jackson ImmunoResearch Laboratories, West
Grove, PA) was added, and plates were incubated at room temperature for
1 h. Following washing, 100 µl of FSAP (diflusinal phosphate,
1/10 in substrate buffer) were added, and plates were incubated for an
additional 10 min at room temperature with shaking. Plates were read by
Cyber Fluor (Beckman Instruments, Fullerton, CA). Units were assayed by
comparison to a mouse TNF- standard (Genzyme).
IL-1.
MHV-3 (at a MOI of 2.5)- or LPS (10 µg/ml)-stimulated macrophages
were incubated with ribavirin (0–500 µg/ml) for 8 or 4 h,
respectively. Production of IL-1ß in supernatants was determined by
ELISA (R&D Systems, Minneapolis, MN). An affinity-purified polyclonal
Ab specific for mouse IL-1ß was precoated onto the microtiter plate.
Standards, controls, and samples were added to the wells for 2 h
at room temperature. After washing, an enzyme-linked polyclonal Ab
specific for mouse IL-1ß was added to the wells, and plates were
incubated at room temperature for 2 h. Following washing, a
substrate solution was added to the wells. After 30 min of incubation
at room temperature, the stop solution was added, and plates were read
by Titertek Multiskan MCC/340 Mk. Ribavirin was added to the
supernatants from MHV-3- or LPS-infected macrophages at a final
concentrations of 1–500 µg/ml before assay, no effects were found on
the ELISA assay for TNF- and IL-1ß.
IL-4 and IFN-.
Production of IL-4 and IFN- in the T cell lines (9) was measured by
bioassay. For the Th1/Th2 cell line, 2 x 104
cells were first stimulated with peritoneal cells from B10BR (C3H) and
cultured with ribavirin (0–500 µg/ml) for 24 h in a final
volume of 200 µl in RPMI 1640 supplemented with 10% FCS, 2 mM
glutamine, 100 U of penicillin, and 100 µg of streptomycin (Flow
Laboratories) per ml in 96-well flat-bottom microtest plates (Falcon
Laboratories, Grand Island, NY). For the MHV-3-specific Th1 (3E9.1) and
Th2 (4B6.8) cell lines, the lines were cocultured in growth factor-free
medium with MHV-3-infected, irradiated splenic mononuclear cells as
APCs, and the supernatants were collected 48 h later. Supernatants
were collected for measurement of IFN- and IL-4 production by the
ability of supernatants to inhibit or support the proliferation of Wehi
279 and CT4.S cells, respectively. Cultures were incubated for 40
h in a humidified CO2 atmosphere at 37°C, pulsed with 1
µCi of [3H]TdR (sp. act. 2 Ci/mmol; Amersham, Arlington
Heights, IL), and harvested 26 h later onto glass fiber filters.
Total cell-associated radioactivity was measured in a Beckman
scintillation counter, and bioassay data were expressed as nanograms
per milliliter for IFN- and units per ml for IL-4, derived from a
standard curve with mouse recombinant IFN- and IL-4 (Genzyme).
Ribavirin was added to Wehi 279 and CT4.S cells at different
concentrations (1–500 µg/ml), showing no effect on the assay system
(data not shown).
Northern (RNA) blot analysis
The effect of ribavirin on mRNA levels of the MHV-3-induced PCA,
fgl2 prothrombinase, TNF, and IL-1 were assayed by Northern blot
analysis. Macrophages (1 x 107) were pelleted in
1.5-ml Eppendorf tubes, and total cellular RNA was isolated by 8 M
acid-guanidium hydrochloride extraction in a modified procedure
described by Arnstein and Cox (10). RNA was resolved on an 1% agarose
gel containing formaldehyde and transferred onto a nitrocellulose
membrane (Bio-Rad, Oakville, Ontario, Canada). A 1.3-kb fgl2
prothrombinase cDNA (11), a 1.4-kb TNF cDNA (12), and a 2-kb IL-1 cDNA
(13) were excised from plasmids, purified by agarose gel
electrophoresis, and labeled by using a random-priming DNA labeling
system (Pharmacia, Montreal, Quebec, Canada) with
[-32P]dCTP (sp. act. 3000 Ci/mmol; Amersham).
Membranes were prehybridized for 5 h at 42°C in a mixture
containing formamide (50%), 5 x Denhardt’s solution, 0.2% SDS,
100 µg of denatured salmon sperm DNA per ml, and 5x SSPE.
Hybridization was conducted overnight at 42°C in the same fresh
mixture. The membranes were then washed under medium to high stringency
conditions, and the membranes were exposed to Kodak XAR-5 film with
intensifying screens for 16 to 24 h at -70°C. To confirm that
equivalent amounts of RNA were loaded in all lanes, the membranes were
probed with -tubulin cDNA.
Statistical analysis
Statistical analysis was conducted by Student t test, and a p value of 0.05 or less was considered to be statistically significant. Results were reported as the mean ± SD for at least three separate experiments, each performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Addition of ribavirin up to 500 µg/ml to peritoneal macrophages freshly isolated from BALB/cJ mice had no toxic effects as demonstrated by trypan blue exclusion (>95% viable).
Effect of ribavirin on the growth of MHV-3 in macrophages
The addition of ribavirin to macrophages from BALB/cJ mice
inhibited MHV-3 replication as demonstrated in Figure 1
. The inhibitory effects showed a
dose-dependent pattern over the range 10 to 500 µg/ml. However, in
macrophages, the maximal inhibitory effect of ribavirin was only 1 log,
from 2.40 ± 0.02 x 107 PFU/ml to 1.55
± 0.07 x 106 PFU/ml at a concentration of 100
µg/ml with no further increase at a concentration as high as 500
µg/ml compared with no ribavirin treatment. The inhibition was not
due to toxic effects of ribavirin on cells as demonstrated by trypan
blue exclusion.
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). Effects of ribavirin on functional PCA and transcription of mRNA for fgl-2 in MHV-3-infected macrophages
Macrophages infected with MHV-3 for 8 h demonstrated a
significant rise in PCA (941 ± 80 mU/106 cells)
in comparison with basal values of 63 ± 15 mU/106
cells. With ribavirin treatment over the range 100 to 500 µg/ml,
macrophages PCA decreased significantly from 941 ± 80
mU/106 cells to 34 ± 11 mU/106 cells
(maximum 94% inhibition) (p < 0.001). The
inhibitory effect of ribavirin showed a concentration-dependent manner
(Fig. 2). To determine whether ribavirin
inhibited the one-stage clotting assay, ribavirin (500 µg/ml) was
added to MHV-3-infected macrophages just before the PCA assay.
Ribavirin had no inhibitory effect on the ability to determine PCA
(data not shown).
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Macrophages produced significantly higher levels of TNF- in
response to MHV-3 infection (10.73 ± 2.15 ng/ml) in comparison
with the basal values (0.03 ± 0.03 ng/ml). Ribavirin
significantly inhibited the production of TNF- in MHV-3-infected
macrophages over the range 100 to 500 µg/ml. The inhibitory effect
showed a dose-dependent pattern with a maximum 75% inhibition in
TNF- (10.73 ± 2.15 to 2.74 ± 0.93 ng/ml)
(p < 0.001) (Fig. 4). By Northern blot analysis, addition
of ribavirin prevented increased levels of TNF mRNA transcription seen
in response to MHV. The reduced effect was first detected at a
concentration of 100 µg/ml with complete inhibition at 500 µg/ml
(Fig. 5A).
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Supernatants from macrophages were analyzed for production of
IL-1. The dose-dependent inhibitory effect of ribavirin on the IL-1ß
production of MHV-3-stimulated macrophages is depicted in Figure 6. Ribavirin significantly inhibited
IL-1ß production from 155.91 ± 22.62 to 5.74 ± 0.70 pg/ml
over the range 10 to 500 µg/ml (maximum 96% inhibition)
(p < 0.001). This inhibitory effect was at the
level of gene transcription as evidenced by Northern blot analysis
(Fig. 5B). The mRNA concentration for IL-1 was
inhibited by ribavirin (100 µg/ml) and was undetectable when cells
were treated with 500 µg/ml ribavirin.
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and IL-1
in LPS-stimulated macrophages even when added at concentrations up to
500 µg/ml (Fig. 7).
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In vitro.
To determine whether ribavirin differentially affected production of
Th1/Th2 cytokines, ribavirin (1–500 µg/ml) was added to the T cell
lines. Supernatants were collected and assayed for the production of
IFN- and IL-4 as described above. Ribavirin, when added at
concentrations from 1 to 500 µg/ml, had no inhibitory effect on the
production of IFN- (84.5 ± 3.7 ng/ml) by either the Th1/Th2
line or the Th1 cell line 3E9.1, as shown in Table I. Addition of ribavirin at a
concentration higher than 100 µg/ml inhibited the production of IL-4
by both the Th1/Th2 line and the Th2 line 4B6.8 (Table I). A
maximum 97% inhibition (p < 0.01) was seen at
concentrations of ribavirin 
100 µg/ml and was not due to toxic
effect of ribavirin on Th2 cells as shown by trypan blue exclusion.
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in
vivo, mice fully susceptible (BALB/cJ), semisusceptible (C3H/HeJ), and
resistant (A/J) to MHV-3 were treated daily with either ribavirin at a
dose of 75 mg/kg/day i.p. or saline beginning 2 h before infection
with 100 PFU of MHV-3. Groups of mice (n = 4)
were sacrificed daily, and sera were collected and assayed for IL-4 and
IFN-. Similar to previous results (21), sera from MHV-3-infected A/J
mice had an increase in levels of IFN-, but no increase in levels of
IL-4 consistent with a Th1 response whereas sera from MHV-3-infected
BALB/cJ mice had an increase in IL-4, but no increase in IFN-
consistent with a Th2 response. Sera from MHV-3-infected
semisusceptible C3H mice had a cytokine profile similar to that of
susceptible BALB/cJ mice. Treatment of mice with ribavirin resulted in
a marked inhibition in levels of IL-4 in semisusceptible and
susceptible mice, whereas it had little effect on production of IFN-
in resistant mice similar to the observed effects in vitro in the Th1,
Th2, and Th1/Th2 lines (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data confirm that although ribavirin has minimal (<1 log)
inhibitory effects on replication of MHV-3 in vitro, even at very high
concentrations complete eradication of the virus was not seen, as has
been previously reported (1, 25, 26). However, ribavirin at
concentrations that are achieved in vivo (4) almost totally inhibited
the production of proinflammatory mediators TNF, IL-1, and PCA in
BALB/cJ macrophages in vitro. Ribavirin was unable to cause similar
inhibition of LPS-induced inflammatory cytokines. This may be due to
the fact that induction of TNF, IL-1, and PCA is due to LPS
induction of IFN which is not inhibited by ribavirin
(27, 28).
In several other animal models of liver injury, including those due to virus infection, endotoxin, CCl4, galactosamine, and acetaminophen, the hepatic injury is associated with fibrin deposition, sinusoidal thrombosis, and accumulation of the inflammatory cells (29, 30, 31, 32, 33, 34, 35, 36, 37). In the hepatocellular necrosis associated with these pathologic processes, resident macrophages within the liver (Kupffer cells) exhibit morphologic features of activation and release a number of inflammatory mediators, including TNF, IL-1, proteolytic enzymes, and eicosanoids, as well as superoxide anions and nitric oxide (36). Furthermore, Chisari (38), using a hepatitis B surface Ag transgenic mouse model, has shown that although CD8+ CTL initiates hepatocyte injury, macrophages and their inflammatory mediators, in particular IL-1 and TNF, are responsible for massive hepatic necrosis. Inactivation of these macrophages prevents hepatic necrosis.
Macrophages generate a wide range of mediator molecules which may contribute either directly or indirectly to the development of fulminant hepatitis by inducing PCA (37). TNF and IL-1 production by macrophages can stimulate endothelial cell production of immune coagulants and increase neutrophil-endothelial interaction, thereby potentially promoting microvascular thrombosis (39, 40).
The immune system plays an essential role in the outcome of viral
infection. One mechanism of the immune response in vivo involves
CD4+ Th cells, which, through the production of cytokines,
control the development of immune effector mechanisms such as Ab
production, generation of cytotoxic T cells, and macrophage activation.
Following Ag exposure, Ag-specific Th cells differentiate along two
pathways (41). Th1 cells, through the production of IL-2, IFN-, and
lymphotoxin, mediate cellular immunity, which is essential for
clearance of viral and other pathogens (42, 43, 44, 45, 46, 47, 48, 49, 50, 51). Th2 cells, conversely,
produce IL-4, IL-10, and IL-13 and are most effective in providing help
for B cell differentiation to plasma cells. IFN- production by Th1
cells inhibits the proliferation of Th2 cells (43), whereas treatment
with neutralizing Abs to IFN- promotes the development of a Th2
response (44, 45).
Our data also showed that ribavirin diminished IL-4 production both by
the Th1/Th2 line and the MHV-3-specific Th2 cell line 4B6.8, while no
effect was seen on IFN- production by Th1 cells, thus preventing the
shift to a Th2 response.
In vivo, treatment of MHV-3-infected semisusceptible C3H/HeJ and
susceptible BALB/cJ mice with ribavirin resulted in inhibition of IL-4
production, but similar doses of ribavirin did not inhibit production
of IFN- production in resistant A/J mice. This may explain the
benefit of ribavirin on amelioration of MHV-3 infection and the
increased survival in vivo previously reported (4).
A Th1 response has been associated with host resistance, and a Th2 response has been associated with susceptibility in murine models of leishmaniasis, candidiasis, listeriosis, and MHV-3 (46, 47). Recently, in acute and chronic hepatitis B virus infection in humans, a Th1 response has also been associated with resistance and a Th2 response with susceptibility (38, 48). Furthermore, immunomodulatory treatments that have converted a Th1 to a Th2 response have resulted in the loss of resistance (38), and, conversely, induction of a Th1 response has led to resolution of the infection (48). In hepatitis C, studies on cytokine profiles have been reported only in patients with chronic disease in contrast to the model of acute fulminant hepatitis induced by MHV-3. The reports in patients with HCV are somewhat conflicting but suggest a poor or absent Th1 response in these patients (49, 50, 51). Recent studies have shown that ribavirin has beneficial effects on serum aminotransferase concentrations and necroinflammatory activity of liver biopsies in patients with chronic HCV infection. However, ribavirin has no effect on serum HCV RNA titers, and biochemical relapse is universal after cessation of therapy (15, 16, 17). The discordance between the virologic and biochemical responses in patients receiving ribavirin suggests that the beneficial effect of ribavirin may not be mediated by inhibition of viral replication. Another possibility is that ribavirin modulates the immune response to HCV.
In conclusion, our study suggests that ribavirin is a very potent
inhibitor of viral-induced proinflammatory mediators. The beneficial
effects of ribavirin in both experimental animal models and clinical
treatment may be related to its ability to markedly reduce macrophage
activation and diminish Th2 cytokine production while preserving Th1
cytokine production. These data provide a rational potential clinical
utility of ribavirin either alone or in combination with IFN- in
patients with hepatitis or as a substitute in IFN--nonresponsive
patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gary Levy, 621 University Avenue, 10th Floor, Room 151, Toronto, Ontario, M5G 2C4, E-mail address:
3 Abbreviations used in this paper: MHV-3, mouse hepatitis virus strain 3; PCA, procoagulant activity; musfiblp, mouse fibrinogen-like protein; HCV, hepatitis C virus; PFU, plaque-forming units; MOI, multiplicity of infection.
Received for publication April 2, 1997. Accepted for publication December 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis. J. Immunol. 147:3149.[Abstract]
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 D. G. Bausch, P. E. Rollin, A. H. Demby, M. Coulibaly, J. Kanu, A. S. Conteh, K. D. Wagoner, L. K. McMullan, M. D. Bowen, C. J. Peters, et al. Diagnosis and Clinical Virology of Lassa Fever as Evaluated by Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent-Antibody Test, and Virus Isolation J. Clin. Microbiol., July 1, 2000; 38(7): 2670 - 2677. [Abstract] [Full Text]
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R. C. Tam, K. Ramasamy, J. Bard, B. Pai, C. Lim, and D. R. Averett The Ribavirin Analog ICN 17261 Demonstrates Reduced Toxicity and Antiviral Effects with Retention of both Immunomodulatory Activity and Reduction of Hepatitis-Induced Serum Alanine Aminotransferase Levels Antimicrob. Agents Chemother., May 1, 2000; 44(5): 1276 - 1283. [Abstract] [Full Text]
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 G. A. Levy, M. Liu, J. Ding, S. Yuwaraj, J. Leibowitz, P. A. Marsden, Q. Ning, A. Kovalinka, and M. J. Phillips Molecular and Functional Analysis of the Human Prothrombinase Gene (HFGL2) and Its Role in Viral Hepatitis Am. J. Pathol., April 1, 2000; 156(4): 1217 - 1225. [Abstract] [Full Text] [PDF]
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I. Jordan, T. Briese, D. R. Averett, and W. I. Lipkin Inhibition of Borna Disease Virus Replication by Ribavirin J. Virol., September 1, 1999; 73(9): 7903 - 7906. [Abstract] [Full Text]
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Q. Ning, M. Liu, P. Kongkham, M. M. C. Lai, P. A. Marsden, J. Tseng, B. Pereira, M. Belyavskyi, J. Leibowitz, M. J. Phillips, et al. The Nucleocapsid Protein of Murine Hepatitis Virus Type 3 Induces Transcription of the Novel fgl2 Prothrombinase Gene J. Biol. Chem., April 9, 1999; 274(15): 9930 - 9936. [Abstract] [Full Text] [PDF]
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 J. G. McHutchison, S. C. Gordon, E. R. Schiff, M. L. Shiffman, W. M. Lee, V. K. Rustgi, Z. D. Goodman, M.-H. Ling, S. Cort, J. K. Albrecht, et al. Interferon Alfa-2b Alone or in Combination with Ribavirin as Initial Treatment for Chronic Hepatitis C N. Engl. J. Med., November 19, 1998; 339(21): 1485 - 1492. [Abstract] [Full Text] [PDF]
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T. J. Liang Combination Therapy for Hepatitis C Infection N. Engl. J. Med., November 19, 1998; 339(21): 1549 - 1550. [Full Text]
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M. Pope, P. A. Marsden, E. Cole, S. Sloan, L. S. Fung, Q. Ning, J. W. Ding, J. L. Leibowitz, M. J. Phillips, and G. A. Levy Resistance to Murine Hepatitis Virus Strain 3 Is Dependent on Production of Nitric Oxide J. Virol., September 1, 1998; 72(9): 7084 - 7090. [Abstract] [Full Text] [PDF]
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