Full text of DOI:10.1089/10799900152434420

JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 21:539–545 (2001)
Mary Ann Liebert, Inc.
Efficacy and Safety of Orally/Sublingually, Intranasally, and
Intraperitoneally Administered Recombinant Murine Interferon
in the Treatment of Murine Encephalomyocarditis Virus
GERALD SONNENFELD,1,2 MICHAEL TOVEY,3 HUUB SCHELLEKENS,4 KEVIN S. KINNEY,1,2
TESFAYE BELAY,1 DARLA S. MORTON,2 CATHERINE E. AUSTIN,2 MARTHA REITMAN,5
T. ANNIE T. FONG,5 and HOLLI S. VAUGHAN6
ABSTRACT
Interferons (IFN) have been shown to be effective in protecting animals against lethal viral infections when
administered systemically in relatively high doses. Intraperitoneal (i.p.) injection of mice with encephalomyo-
carditis virus (EMCV) gives rise to a rapidly progressive fatal disease characterized by central nervous sys-
a
tem involvement and encephalitis. IFN- has been shown to be effective in protecting mice against lethal
EMCV infection when given via parenteral and oral/sublingual routes. The current study was designed to ex-
a
plore the ability of orally/sublingually and intranasally (i.n.) administered IFN- to treat mice infected with
a
EMCV in support of a planned clinical trial to evaluate efficacy of oral IFN- in human viral infections. The
a
a
primary objective of the study was to determine the efficacy of recombinant murine IFN- (rMuIFN- ) in
the treatment of mice infected with 100 LD50 EMCV following oral, i.n., and i.p. administration at doses of
20,000 and 100,000 IU. The results of the current experiment did not indicate protection from infection with
EMCV in mice that received IFN by the i.n. or oral/sublingual routes. The negative controls, infection of mice
with 100 LD50 of EMCV followed by treatment with excipient via all three routes, resulted in death of nearly
a
all mice, as expected. The positive control, treatment of EMCV-infected (100 LD50) mice with rMuIFN- via
the i.p. route, was successful in protecting a significant number of mice from death compared with matched
controls. This study points out the need to determine the optimum conditions for administration of oral/sub-
lingual or i.n. IFN to insure maximum efficacy against viral infections.
of administration of IFN via the oral/sublingual or intranasal
INTRODUCTION
(i.n.) routes in protecting animals from a wide variety of viral
(5)
NTERFERONS (IFN) ARE AMONG THE CYTOKINES most widely infections. These results were compiled and summarized in
Journal of Interferon and Cytokine
A number of studies have failed to detect biolog-
used in clinical medicine today, with applicationsboth in on- a recent special issue of the
I
(1)
Research (5)
cology and in the treatment of viral infections. Although a
number of routes of IFN administration, including intravenous
(i.v.),subcutaneous(s.c.),intramuscular(i.m.), and intralesional
(i.l.), are commonly used, the oral route rarely has been used
.
ically active IFN in the blood following oral administration,
(2–4)
suggesting the possibility of novel mechanisms of action.
IFN have been shown to be effective in protecting animals
clinically because IFN are proteins that are inactivated by di- against lethal viral infections when administered systemically
gestive tract proteolytic enzymes, such as trypsin. In addition, in relativelyhigh doses.(6) Intraperitonealinjectionof mice with
IFN are not absorbedappreciablyin their native form.(2–4) Nev- encephalomyocarditis virus (EMCV) gives rise to a rapidly pro-
ertheless, multiple reports have demonstrated the effectiveness
gressive fatal disease characterized by central nervous system
1Department of Microbiology Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA 30310-1495.
2Department of General Surgery Research, Carolinas Medical Center, Charlotte, NC 28232.
3Laboratory of Viral Oncology, CNRS, Villejuif, France.
4Medical Microbiology, RU Utrecht, The Netherlands.
5Reitman Corporation, Palo Alto, CA 94306.
6Pharma Pacific Pty. Ltd., Washington, DC 22206.
539

540
SONNENFELD ET AL.
(7)
activity was measured by the cytopathic effect inhibition assay
on mouse L-929 cells challengedwith Semliki Forest virus. The
solution was filter sterilized. International units (IU) were cal-
ibrated against the NIH laboratory standard for MuIFN-a/b
(Ga02-901-511). Individual aliquots of rMuIFN-a were stored
at 270 to 280°C. Phosphate-bufferedsaline with bovineserum
albumin (PBS/BSA, 0.01%) was prepared in the laboratory and
was obtained from Sigma Chemical Co. (St. Louis, MO). The
rMuIFN-a was diluted in PBS/BSA. EMCV (200 ml) obtained
from the American Type Culture Collection (ATCC VR 129)
(Rockville, MD) was injected i.p. into each mouse.
(CNS) involvement and encephalitis. IFN-a has been shown
to be effective in protecting mice against lethal EMCV infec-
tion.(6) Intranasal or oral/sublingual administration (or both) of
IFN-a has been shown to successfully protect mice infected
with EMCV.(8)
The current study was designed to confirm that oral/sublin-
gual and i.n. IFN-a treatment protected mice infected with
EMCV in support of a planned clinical trial to determine effi-
cacy of oral/sublingual IFN-a in a human viral infection. The
study consisted of two parts, the EMCV titration and the anti-
viral assessment. The objective of the EMCV titration was to
determine the dose of EMCV required to kill 50% of the in-
fected animals (LD50). The primary objects of the antiviral ac-
tivity assessment was to determine the efficacy of recombinant
murine IFN-a (rMuIFN-a) in the treatment of mice infected
with 100 LD50 of EMCV following oral/sublingual, i.n., and
i.p. administrationat doses of 20,000 and 100,000 IU rMuIFN-
a. The primary measure of effect was defined as survival at 21
days, and the primary efficacy analysis was the relative survival
at 21 days of each treatment group in comparison to its control
group, which received only excipient.
In addition to the principal objectives,the study also was de-
signed to (1) provide an additionalevaluationof efficacy,where
time to death following infection was measured, and (2) eval-
uate the safety of rMuIFN-a in the treatment of mice infected
with 100 LD50 of EMCV following oral/sublingual, i.n., and
i.p. administration at doses of 20,000 and 100,000 IU. Safety
was evaluated both by clinical assessment and by gross patho-
logic assessment.
Administration of IFN
The first dose of IFN was given 1–2 h after inoculationwith
100 3 LD50 EMCV. Dosing always began in the morning and
continued until the last animal scheduled to be dosed that day
was dosed. The treatment groups were always dosed in ap-
proximately the same order so that dosing occurred at approx-
imately the same time for each treatment group. Because of the
unexpected loss of material during administration, only
80%–90% of the designated IFN dose was administered on day
1. Four mice in the low-dose (20,000 IU) i.n. IFN 1 EMCV
group and 11 mice in the high-dose (100,000 IU) i.n. IFN 1
EMCV group mice did not receive any IFN on day 1 of the ex-
periment. All mice received 100% of the designated IFN dose
on days 2–4 of the experiment. Intranasal IFN was adminis-
tered in a total volume of 10 ml—5 ml to each nostril. Oral/sub-
lingual administrationwas carried out by placing 10 ml IFN un-
der the tongue of the mouse. Additional IFN was administered
in a volume of 200 ml via the i.p. route. PBS/BSA excipient
was administered in equivalent volumes via each route to neg-
ative control animals.
MATERIALS AND METHODS
Animals
Study design
EMCV titration
. Groups of 12 female (approximately48 days
Specific pathogen-free female Swiss mice were obtained
from Charles River Laboratories (Wilmington, MA). All pro- old) Swiss mice were uninfected and received i.p. 200 ml PBS
cedures were approved by the Institutional Animal Care and only or were infected i.p. with 200 ml PBS containing 10-fold
Use Committee of the Carolinas Medical Center. The mice were serial dilutions of EMCV ranging from 1022 to 1027. After in-
approximately 5 weeks old and weighed approximately 14–20 fection,the mice initially were observedfour times daily, within
g when received for the EMCV titration. The mice were ap-
an hour at 07:00, 12:00, 17:00, and 21:00, and any deaths were
proximately 9 weeks old and weighed approximately 23–27 g noted. The number of daily observations was reduced to two
when received for the antiviral assessment. The increase in (08:00and 16:00)when it appearedthat no additionalmice were
weight was necessitated by the availability of mice from the expected to die. Day of death was defined as a 24-h day be-
supplier. On receipt, the animals were put into quarantine for ginning at the time of EMCV inoculationfor each animal (e.g.,
7 days. After 7 days of quarantine, the mice were retained in 09:00). A death that occurred between 09:00 on day 3 and up
the study room.
to 09:00 on day 4 was credited to day 3. Animals that were ob-
served to be in distress and near death, as evidenced by ruffling
of fur and moribund and inactive behavior, were immediately
killed by cervical dislocation to minimize pain and suffering
and were counted as dead animals. The animals were observed
for 21 days, well beyond the time anticipated for EMCV in-
fection to inducemortality.A Reed-Muenchanalysis(9) was car-
ried out to determine the 50% end point of death, the LD50.
Randomization
Groups of 44 mice were used to ensure sufficient power for
appropriate statistical analysis. Mice were selected randomly
and assigned sequentiallyto route: oral/sublingual, i.n., i.p., ex-
cipient only, safety only, or sentinel. IFN-treated groups were
then assigned sequentially to dose (20,000 IU or 100,000 IU).
Antiviralassessment
. To maximize dose-ranginginformation
by route and to minimize the number of animals required,
oral/sublingual,i.n., and i.p. routes were evaluated in two doses
Reagents
rMuIFN-a was obtained from PBL Biomedical Laboratories (20,000 IU and 100,000 IU) to allow for comparison. The ex-
(New Brunswick, NJ; lot number 1094). The rMuIFN-a was
determined to be .95% pure by affinity chromatography.Unit served as a positive control. The safety only group was ad-
cipient only group served as a negative control. The i.p. route

541
ORALLY ADMINISTERED IFN AND EMCV
ministered EMCV and served as a control for evaluation of for any deaths following EMCV inoculation, 44 animals were
safety in comparisonwith the EMCV treatmentgroupsin which allocatedto each group.Death countedtoward the efficacyanal-
there was also drug treatment. The sentinel group served as a ysis if the animal had received at least one dose of IFN-a.
control for changes in environmental conditions.
The rMuIFN-a was administered once a day for 4 days. The
first dose was given 1–2 h after inoculation with 100 3 LD50
EMCV, the dose used in the successful preliminary study.(8)
RESULTS
Dosing always began in the morning and continued until the
last animal scheduledto be dosed that day was dosed. The treat-
LD50 determination
ment groups were always dosed in approximately the same or-
der so that dosing occurred at approximately the same time for randomly assigned to groups of 12 mice each. One group re-
each treatment group. ceived 200 ml PBS i.p. Six other groups of 12 mice each re-
Female Swiss mice, after quarantine and acclimation, were
Clinical assessments for all animals were conducted after ceived 10-fold serial dilutions of EMCV in 200 ml PBS, yield-
they were released from quarantine, and the findings were ing doses of 1022–1027 dilutionsof virus. The titer of the virus
recorded. These observations included but were not limited to was determined to be 6.5 3 106 plaque-forming units (pfu)/ml
changes in skin and fur, eyes and mucous membranes, respira- by plaque assay on L-929 cells. Mice were observed for 21
tory system, circulatory system, autonomic nervous system and days, four times daily for 14 days, and then twice daily for the
CNS, somatomotor activity, and behavior pattern.
last 7 days, when it became apparent that deaths from viral in-
Mice were observed four times daily through day 21 fection had ended. Animals that showed signs of paralysiswere
postinoculation, and any deaths were noted. Observation con- killed by cervical dislocation. Deaths ended by day 5 after in-
ditions were similar to those described for the LD50. Compar- fection (data not shown). The LD50 for the mice was calculated
isons were made between the oral/sublingual and i.n. treatment by means of a Reed-Muench estimation and was determined to
groups and their respective excipient only group for survival. be 1025.6 dilution of viral stock (3.3 viral particles in 200 ml
The animals were monitoredfor survivalfor a periodthrough PBS). Based on the previous studies of Tovey and Maury,(8)
a
21 days after EMCV administration.Animals surviving21 days concentration of 100 LD50 of EMCV was used for future ex-
of the study were killed on day 21 after the 08:00 observation periments. As the initial titer of virus was 6.5 3 106 pfu/ml,
using cervical dislocation.
this was obtained by diluting the stock virus by 1:3981.
IFN titration
Statistical analysis
The IFN assay was based on a cytopathic effect using the
Indiana strain of vesicular stomatitis virus (VSV). The assay
was carried out in 96-well flat-bottomed microtiter plates. An
internationalreferencestandard for rMuIFN-a (Ga02-901-511)
was obtained from the NIH/WHO and used to calibrate the as-
say. An internal laboratory standard was prepared from a com-
merciallyobtainedpreparationof purified MuIFN-a/b (Sigma).
This laboratory standard, calibrated in our assay against the in-
ternational reference standard, was found to contain 33% of the
expected IU. This standard was run with all further assays, and
all values were multiplied by 3 to obtain IU values.
Commercial IFN was obtained from PBL Biomedical Lab-
oratories and contained glycerol (MuIFN-a A, lot 1094, spec-
ified titer 1 3 107 IU/ml). In our assay, the titer was 64.8 3
107 IU/ml, substantiallyhigher than specified. The IFN was as-
sayed multiple times by different individuals, and similar re-
sults were obtained. The IFN was stored at 270°C.
Standard statistical methods were used. Descriptive statis-
tics, including means and standard deviations (SD) or counts
and percentages, were calculated. The principal analysis com-
pared survival at day 21 in each group with its respective ex-
cipient only group (virus and no IFN). The groups differed by
dose level of IFN-a and the route of administration. The one-
tailed test was used, as the alternative hypothesisis that the sur-
vival rate will be higher than in the excipient only group (the
mortality in the excipient only group was near 100%).
A secondary analysis compared the time to death in each
group comparedwith its respectiveexcipientonlygroup.Kaplan-
Meier survival curves were plotted, and the log-rank statistic
was tested for differences among the groups. The gross pathol-
ogy on each mouse was characterized as normal or abnormal.
Safety was assessed through an evaluation of observation and
gross pathology. Data were tabulated, and descriptivestatistics,
including means and SD or counts and percentages, were cal-
culated.
Antiviral efficacy experiment
p
The SAS software was used for all analyses. A value of
#0.05 was considered statistically significant. The expected
The animals were infected with virus and treated with IFN
percent survival at day 21 was ,5% in the controls and 25% by the study director and research assistants. Treatments were
in the i.p. IFN-a treatment groups, based on previous work by staggered, and all animals were treated with IFN beginning 1
Tovey and Maury.(8)
h after and completed within 2 h after viral infection. Because
The sample size was based on the principal comparison of titration of the rMuIFN-a indicated a titer substantially greater
the percent surviving on day 21 of each group with the re- than specified, a question arose as to which dose should be ap-
spective control group. A one-tailed chi-square test requires 39 plied. Two alternatives were considered. One was to apply the
animals per group to achieve a power of 80%, with an alpha IFN assuming the dose to be the same as specified by the sup-
level of 0.05. This calculationalso assumes that the percent sur- plier (PBL). The other was to apply the IFN assuming the dose
viving in the control group is 5% and the clinical difference is indicated by the laboratory assay. If the PBL titer were used,
20% (i.e., at least a 25% survival in the other group). To allow the risk was that the actual dose would be so high that it could

542
SONNENFELD ET AL.
TABLE 1. STATISTICAL ANALYSIS OF TREATMENT VS. CONTROLS (ALL ANIMALS)
Treatment group
% Survivala
Control % survival
value
p
High-dose i.p. IFN
Low-dose i.p. IFN
High-dose i.n. IFN
Low-dose i.n. IFN
High-dose sublingual/oral IFN
Low-dose sublingual/oral IFN
26/44, 59.1%b
32/44, 72.7%b
1/44, 2.3%
3/44, 6.8%
1/44, 2.3%
4/44, 9.1%
5/44, 11.4%
5/44, 11.4%
1/44, 2.3%0
1/44, 2.3%0
3/44, 6.8%0
3/44, 6.8%0
,0.001
,0.001
NSc
NSc
NSc
NSc
aComparing different treatment groups with their appropriate control group.
bThe survival rate for the group given high-dose IFN via the i.p. route was compared with the group given low-dose IFN via
the i.p. route. The results were not statistically significant.
cNS, not significant.
be toxic to the mice. If the laboratory titer were used, the risk it was impossible to provide the full dose of IFN on the first
was that the actual dose would be so low that any potentialben- day. The IFN-treated mice received between 80% and 90% of
efit would be lost because of dilution of the IFN. A decision the intended dose. This was likely to have minimal impact be-
was made to treat two extra test mice with 100,000 U IFN based cause, if anything, the actual IFN titer was higher than the PBL
on the PBL specified titer. One mouse was treated via the i.p. titer used to calculate the dose. Even the extreme measures
route, and the other was treated via the oral/sublingual route. taken did not allow us to provide a full dose to all mice that re-
The mice were observed for 24 h. No obvious toxicity was ob- ceived IFN by the i.n. route. Four mice that received low-dose
served, so we used doses specified by the manufacturer. The (20,000 IU) IFN via the i.n. route 1 EMCV and 11 mice that
decision was made that the risk of diluting the IFN too much received high-dose (100,000 IU) IFN via the i.n. route 1
was greater than the risk of toxicity. Reports have indicated EMCV did not receive any IFN on day 1 of the experiment.
(10)
considerable variability in IFN assays,
problem.
and this remains a Because we were aware of this difficulty, we were able to com-
pensate for it on the following days. All mice received the full
After dilutions were made with PBS/BSA, it became clear dose of IFN on days 2–4 of the experiment.
that IFN was being lost in the hubs of the syringes and on the
Several of the mice that received IFN via the i.n. route
sides of the tubes holding the IFN for the i.n. and oral/sublin- showed signs of lethargy for about 60 sec after administration.
gual applications. It was noted that the IFN samples were very They recoveredrapidlyand showed normal behaviorwithin 2–3
viscous,and it is possiblethat the glycerolcould have accounted min after dosing. The i.n. dose may have momentarily inter-
for this unexpectedloss. As the dilution had already been made, fered with breathing of the mice. The four times daily clinical
FIG. 1. Survival plot after IFN treatment via the i.p. route. All mice received EMCV.

543
ORALLY ADMINISTERED IFN AND EMCV
FIG. 2. Survival plot after IFN treatment via the oral/sublingual route. All mice received EMCV.
assessmentshowed no untoward signs other than occasionaleye statistical significance for the differences in total survival for
irritation and bleeding from the mouth. This did not appear to the mice that received IFN i.p. compared with appropriatecon-
be linked to the treatment but rather to grooming and behav- trols but no statistically significant difference in survival be-
ioral interactionsof the mice. All mice remained grossly healthy tween oral/sublingualor i.n. IFN-treated mice and matched con-
until paralysis and death began as a result of the EMCV infec- trols (Table 1 and Figs. 1, 2, and 3). Mean survival time was
tion. All control mice remained grossly clinically healthy enhancedfor mice that received IFN via the i.p. route compared
p
with matched controls ( , 0.001) but not between mice that
throughout the experiment until they were killed.
The results of the treatment are shown in Table 1. There was received IFN via the oral/sublingualor i.n. route compared with
FIG. 3. Survival plot after IFN treatment via the i.n. route. All mice received EMCV.

544
SONNENFELD ET AL.
TABLE 2. STATISTICAL ANALYSIS OF TREATMENT VS. CONTROLS (WITHOUT ANIMALS NOT GIVEN IFN ON DAY 1)
Treatment group
% Survival
Control % survival
value
p
High-dose i.p. IFN
Low-dose i.p. IFN
High-dose i.n. IFN
Low-dose i.n. IFN
High-dose sublingual/oral IFN
Low-dose sublingual/oral IFN
26/44, 59.1%
32/44, 72.7%
1/33, 3.0%
1/40, 2.5%
1/44, 2.3%
4/44, 9.1%
5/44, 11.4%
5/44, 11.4%
1/44, 2.3%0
1/44, 2.3%0
3/44, 6.8%0
3/44, 6.8%0
,0.001
,0.001
NSa
NSa
NSa
NSa
aNS, not significant.
matched controls (Table 1 and Figs. 1, 2, and 3). An analysis infected (100 LD50) mice with IFN via the i.p. route, the pos-
of the data not including mice that had not received IFN on day itive controls, was successful in protecting a significant num-
1 showed that mice that received IFN via the i.p. route were ber of mice from death compared with matched controls. This
protected against EMCV infection but mice that received IFN indicated that it was unlikely that the possible reduced dose of
via the oral/sublingual or i.n. route were not protected (Table IFN received on the first day by most mice contributed to the
2). Therefore, the study was so highly powered that statistical lack of positive effect observed in the oral/sublingual and i.n.
analysis proved that eliminatingmice that had not received IFN groups. Additionally,the study was sufficientlypowered so that
on all days had no influence on the results (Table 2). Mean sur- omission from the statistical analysis of mice that did not re-
vival time of EMCV-infected mice was enhanced by i.p. IFN ceive IFN via the i.n. route on the first treatment day did not
treatment, but IFN treatment by the oral/sublingual and i.n. affect the outcome of the analysis.
routes had no effect (Table 3).
It appears, therefore, that the experimentwas carriedout suc-
No gross pathologic problems beyond those induced by the cessfullywithout major technicalissues. The results of the pres-
(5,8)
virus were noted. After the 21-day 08:00 evaluation period, the ent study do not confirm studies
mice were killed by cervical dislocation. No gross pathologic the same extent.
that were not powered to
signs were observed in the euthanized mice.
The difference in results may be due to an inability to show
The experiment was repeated comparing effects of IFN con- statistically the same trends of the previous studies because of
taining glycerol and IFN without glycerol. No significant dif- the greatly enhanced powering of the present study. However,
ferences were observed between the two types of IFN (data not an alternative explanation could be a difference in the proce-
shown).
dures/reagents used. One possibility was that the glycerol con-
tent of the IFN used in the study interfered with the activity via
the i.n. or oral/sublingual routes. This could have accounted for
the observed high viscosity of the IFN and could have affected
the distributionof small volumes of IFN via the oral/sublingual
DISCUSSION
These results do not indicate protection from infection with and i.n. routes. A repeat of this highly powered study using IFN
EMCV in mice that received IFN by the i.n. or oral/sublingual without glycerol gave similar results, suggesting that glycerol
routes. This is in contrast to previous studies, including one was not the problem.
with EMCV.(5,8)
Several other factors could have been responsible for differ-
The negative controls, mice infected with 100 LD50 of ences in the results between the present study and previous re-
EMCV followed by treatment with PBS/BSA excipient via all ports.(5,8) Such factors include differences in strains of mice, in
three routes, nearly all died, as expected. Treatment of EMCV- the source and makeup of the IFN used, in viral strains, and in
TABLE 3. MEAN SURVIVAL TIME OF EMCV-INFECTED MICE TREATED WITH IFN
Mean survival
Treatment group
time (days)a
valueb
p
High-dose i.p. IFN
Low-dose i.p. IFN
i.p. excipient only
High-dose i.n. IFN
Low-dose i.n. IFN
i.n. excipient only
High-dose sublingual/oral IFN
Low-dose sublingual/oral IFN
Sublingual/oral excipient only
15.25 6 7.20
16.75 6 7.05
6.32 6 5.38
4.93 6 2.77
5.64 6 4.33
4.84 6 2.77
4.86 6 2.56
6.07 6 4.82
5.66 6 4.32
,0.005
,0.005
—
NSc
NSa
—
NSa
NSa
—
a6 SD.
bp
value of treatment compared with matched excipient only control.
cNS, not significant.

545
ORALLY ADMINISTERED IFN AND EMCV
Oral use of interferons and cytokines. J. Interferon Cytokine Res.
19, 813–978.
experimental procedures. The results of the present study indi-
cate that these differences must be identified to allow for the
design of protocols that can assure standardized beneficial ef-
fects of orally/sublingually and i.n. administered IFN.
6. FINTER, N.B. (1973). Interferons and interferon inducers in vivo:
I. antiviral effects in experimental animals. Front. Biol. 2, 295–360.
7. RUECKERT, R.R. (1985). Picornoviruses and their replication. In:
Virology. 1st ed. N.B. Fields (ed.) New York: Raven Press, pp.
705–738.
8. TOVEY, M.G., and MAURY, C. (1999). Oromucosal interferon
therapy: marked antiviral and antitumor activity. J. Interferon Cy-
tokine Res. 19, 145–155.
9. REED, L.J., and MUENCH, H. (1938). A simple method of esti-
mating fifty percent end points. Am. J. Hyg. 27, 493–497.
10. MIRE-SLUIS, A.M., DAS, R.G., MEAGER, A., and THORPE, R.
(1998). International collaborative study for proposed international
standards for interferon alpha. WHO International Laboratory for
Biological Standards, Potters Bar, U.K.
ACKNOWLEDGMENTS
This study was funded in part by a grant from Pharma Pa-
cific Pty., Ltd. The authors thank Dr. James Norton of the Car-
olinas Medical Center for his help in design of the experiments
and for statistical analysis of the data.
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Address reprint requests to:
Dr. Gerald Sonnenfeld
Department of Microbiology, Biochemistry and Immunology
Morehouse School of Medicine
720 Westview Drive, S.W.
Atlanta, GA 30310-1495
Tel: (404) 752-1586
Fax: (404) 752-1179
E-mail:
sonneng@msm.edu
5. BIELHARZ, M.W., and FLEISCHMANN, W.R., JR. (eds). (1999). Received 15 February 2000/Accepted 27 March 2001