Method and compositions for treatment of trauma-associated sepsis with gamma interferon

Trauma-associated sepsis is effectively treated using gamma interferon alone or in combination with an antibiotic.

BACKGROUND 
The present invention relates in general to methods and compositions for 
treatment of trauma-associated sepsis, and in particular to methods and 
compositions including gamma interferon ("IFN-Y.sub..gamma. "), especially 
human IFN-.sub.65 , for treatment of traumaassociated sepsis. 
Infection remains the major cause of late death in trauma patients. 
Antibiotics, sterile techniques, mechanical barriers, and conventional 
treatment have met with limited success in preventing or controlling these 
infections. 
A number of immunological parameters have been shown to be depressed after 
trauma. Expression of HLA-DR antigen (which corresponds to the Ia antigen 
in mouse models) is decreased on monocytes of human trauma patients. 
Viral infections and tumors may be treated using .alpha.-interferon or 
IFN-.sub..gamma. as may intracellular bacterial infections. In a suggested 
mechanism for the effect of IFN-.sub..gamma. on intracellular bacterial 
infections, it is proposed that the IFN-.sub..gamma. prevents uptake of 
the bacteria by cells. This mechanism does not relate to infections which 
are not intracellular in nature. 
SUMMARY OF THE INVENTION 
A method according to the present invention for treatment of 
trauma-associated sepsis, particularly of a bacterial blood infection 
caused by an enteric bacterium, includes the step of administering a 
therapeutically effective dose of IFN-.sub..gamma. to a victim of trauma, 
and in particular, to a trauma patient not known to have a viral 
infection, a tumor or an intracellular bacterial infection. The method 
involves introducing IFN-.sub..gamma. into a bodily fluid of a patient. 
This introduction may be effected by injecting IFN-.sub..gamma. 
subcutaneously, intramuscularly or intravenously. Intravenous 
administration may be by infusion or bolus injection. The IFN-.sub..gamma. 
used according to the present invention is preferably desCysTyrCys human 
IFN-.sub..gamma.. 
A method according to the present invention preferably also involves 
co-administering an antibiotic with the IFN-.sub..gamma.. 
Co-administration may be effected by administration of the antibiotic and 
IFN-.sub..gamma. during the course of the same infection, or may be 
effected by inclusion of the antibiotic and IFN-.sub..gamma. in the same 
composition (particularly where administration is by intravenous 
infusion). 
The antibiotic may be selected from the group consisting of cefazolin, 
nafcillin, vancomycin, cefoxitin, neomycin plus erythromycin, penicillin 
G, trimethoprim plus sulfamethoxazole, and clindamycin or clindamycin plus 
gentamycin or tobramycin. 
A composition for treatment of trauma-associated sepsis according to the 
present invention includes IFN-.sub..gamma., an antibiotic and a diluent 
or carrier. The antibiotic may be selected from the group consisting of 
cefazolin, nafcillin, vancomycin, cefoxitin, neomycin plus erythromycin, 
penicillin G, trimethoprim plus sulfamethoxazole, and clindamycin or 
clindamycin plus gentamycin or tobramycin.

DETAILED DESCRIPTION 
Trauma-associated sepsis or infection remains a serious problem despite 
advances in antibacterial chemotherapy. According to the present 
invention, augmenting host immune defenses with IFN-.sub..gamma. increases 
the frequency of survival of patients suffering from such sepsis or 
infection. 
Trauma-associated sepsis is defined herein as the presence of extracellular 
bacteria in the blood or tissues ("sepsis") associated with a wound or 
injury. Bacteria characteristic of trauma-associated sepsis include 
enteric bacteria, and in particular include coliform bacteria, bacilli of 
the genus Klebsiella and bacilli of the genus Pseudomonas. Trauma includes 
physical injury such as surgical incision. 
Bacteria which are recognized as being associated with trauma include: 
Gram-positive bacteria such as Staphylococcus aureus, Streptococcus 
faecalis, Pneumococci, anhaemolytic Enterococci, Sarcina species and 
haemolytic Streptococci; and Gram-negative bacteria such as Escherichia 
coli, Pseudomonas species, Klebsiella species, Proteus species, 
Enterobacter cloacae, coliform bacteria, Serratia species, Citrobacter 
species and Providencia species [Allgower et al., Surg. Clin. N. Am., 60, 
133-144 (1980)]. 
Thus, trauma-associated sepsis may be distinguished from other forms of 
bacterial infection as being characterized by the presence of trauma 
(i.e., injury, such as a burn or a wound) and the relative predominance of 
bacteria which are not intracellular in location. Sepsis after trauma 
usually occurs within two weeks after injury in patients who are 
immunosuppressed as a result of the trauma. Sepsis may be due to microbial 
contamination at the time of trauma, or to spillover of organisms from the 
gut. 
Human IFN-.sub..gamma. is defined herein as a polypeptide having the 
sequence of native gamma interferon as set forth in European Publication 
No. 77,670 and all amino acid sequence or other variants thereof which are 
capable of activation of monocytes from trauma patients. Examples of such 
variants are alleles or the products of site directed mutagenesis in which 
residues are deleted, inserted or substituted. For example, see European 
Publication No. 146,354. 
IFN-.sub..gamma. according to the present invention may be derived from any 
source, including IFN-.sub..gamma. isolated from natural sources, and 
chemically synthesized or recombinantly-produced IFN-.sub..gamma.. 
IFN-.sub..gamma. should be used which is homologous to the animal species 
to be treated since it may not be active across species lines. In human 
therapy, the desCysTyrCys variant of the sequence shown in EP 77,670 is 
preferably employed, and optionally the C-terminal variant in which the 
last 4 residues are deleted in post-translational processing. 
IFN-.sub..gamma. having native sequences is obtained by purification from 
natural sources using known methods. The same molecule or its variants are 
obtained from recombinant sources, also by known methods. 
A typical formulation may contain IFN-.sub..gamma. (20 X 10.sup.6 U) at 1.0 
or 0.2 mg/ml, succinic acid at 0.27 mg/ml, disodium succinate hexahydrate 
0.73 ml/injection at pH 5.0. This aqueous formulation is administered at 
therapeutic doses, which are less than the maximum tolerated dose in 
humans as determined by the clinician. IFN-.sub..gamma. may also suitably 
administered from a reconstituted lyophilized preparation. 
IFN-.sub..gamma. may be administered by any conventional route that directs 
a therapeutic dose to the site of the trauma, for example, by intravenous 
or intrapulmonary (European Publication No. 257,956) delivery routes. 
Administration may be by continuous infusion or bolus dosing in sufficient 
amounts to maintain therapeutic levels. IFN-.sub..gamma. may be 
administered prior to the trauma, in the case of surgical trauma, or as 
soon as possible following accidental or non-surgical trauma, and then 
continued for a time sufficient to permit proper healing of the trauma, 
typically about from 3 to 14 days or such other period as is determined by 
the clinician. 
IFN-.sub..gamma. is preferably administered according to the present 
invention subcutaneously at doses of from 0.01 to 0.1 mg/m.sup.2 /day for 
7-10 days. However, other routes of administration, such as intramuscular 
or intravenous administration, may be employed. Moreover, immunologically 
effective dosage levels may generally be determined for a particular 
application according to the procedure of Maluish et al., J. Clin. Oncol., 
6, 434-445 (1988). 
According to the present invention, IFN-.sub..gamma. may be administered 
therapeutically (i.e. after appearance of sepsis) or prophylactically 
(i.e., prior to appearance of sepsis). 
IFN-.sub..gamma. antibiotics may be co-administered with synergistic effect 
according to the present invention. Thus, trauma-associated sepsis which 
is not effectively treated by antibiotics alone may be treated according 
to the present invention. Antibiotics which may be useful according to the 
present invention include penicillin, ampicillin, amoxacillin, 
methicillin, oxacillin, cloxacillin, diclosacillin, nafcillin, 
carbenicillin, cephalothin, cefazolin, cephradine, cephalexin, 
erythromycin, lincomycin, clindamycin, chloramphenicol, tetracycline, 
doxycycline, minocycline, gentamicin, kanamycin, tobramycin, vancomycin, 
trimethoprim and sulfamethoxazole. [Cushing, Surg. Clin. N. Am., 57, 
165-177 (1977)] 
The following Examples more particularly illustrate the invention. 
In Example 1, the ability of IFN-.sub..gamma. treatment to alter the course 
of infection is demonstrated in a model of surgical wound infection. This 
model simulates a classical surgical wound infection involving pathogenic 
bacteria, a foreign body, tissue injury and an open wound. Simulation is 
obtained by placing a suture impregnated with Klebsiella pneumoniae ("K. 
pneumoniae") into the thigh of a mouse. Mortality rates are high for 
untreated animals in this model. 
In Example 2, the simulated wound infection-treatment model involves 
subcutaneous injection of IFN-.sub..gamma.. Bacterial challenge in this 
model consists of intramuscular injection of K. pneumoniae. 
In Example 3, an infected burn wound model was used to study the efficacy 
of IFN-.sub..gamma. therapy. Two different Gram-negative wound infections, 
specifically those involving K. pneumoniae and Pseudomonas aeruginosa ("P. 
aeruginosa"), were studied. 
Example 4 includes a description of a laparotomy model of clinical 
conditions following trauma. Bacterial challenge involved intraperitoneal 
injections of Escherichia coli ("E. coli") or intramuscular injection of 
K. pneumoniae. 
Example 6 is a description of a determination of values for MO.sub.2 DR 
(i.e. for monocyte HLA-DR antigen expression) in cultures of blood samples 
from normal subjects and trauma patients before and after incubation with 
IFN-.sub..gamma.. 
Example 5 involves a description of a study of the effect of 
IFN-.sub..gamma. on monocyte HLA-DR antigen expression in trauma patients. 
HLA-DR antigen expression by monocytes and macrophages is necessary for 
effective antigen presentation to lymphocytes. Traumatic injury and sepsis 
reduce HLA-DR antigen expression of monocytes. 
In Example 7, a study is described in which patients with severe injury 
received recombinant IFN-.sub..gamma. therapy along with prophylactic 
antibiotic therapy. 
Example 8 illustrates the increased effectiveness against bacterial 
infection observed with the use of an antibiotic plus IFN-.sub..gamma., as 
opposed to the use of either alone. 
EXAMPLE 1 
Adult male CBA/J mice, weighing 20-25 g each, (Jackson Laboratories, Bar 
Harbor, Maine) were deprived of food but allowed free access to water for 
24 hours prior to bacterial challenge and refed 4 hours following that 
challenge. This model has been shown to parallel infections of surgical 
wounds. Polk et al., Surgery, 90, 376 (1981). 
Lengths of 3-0 twisted cotton suture were incubated overnight in trypticase 
soy broth (BBL Microbiological Systems, Cockeysville, Maryland) and 
inoculated with K. pneumoniae (Capsular Type 2). Lengths of suture 
attached to a French eye needle were inserted aseptically into the right 
thigh of each mouse and the suture was cut flush with the skin at either 
end and buried subcutaneously. 
Murine IFN-.sub..gamma., produced by recombinant DNA technology, was a gift 
of Genentech, Inc., South San Francisco, Calif. The IFN-.sub..gamma. had 
specific activity of approximately 2.3.times.10.sup.7 U/mg protein and was 
diluted with RPMI-1640 medium (Gibco Laboratories, Grand Island, N.Y.). 
The interferon was titered prior to use by means of a microplaque 
reduction assay using the Indiana strain of vesicular stomatitis virus on 
L-929 cells. Campbell et al., Can. J. Microbial., 21, 1247-1253 (1975). In 
this assay, one interferon unit was equivalent to 0.88 NIH G-002-904-511 
reference standard units. 
IFN-.sub..gamma. was administered subcutaneously into the left or opposite 
hind limb in daily 0.1 ml injections of 7500 units each. Treatment 
commenced 5 days prior to suture challenge and the last dose was 
administered 1 hour prior to this challenge. In all experiments, control 
animals received 0.1 ml of RPMI-1640 medium subcutaneously at the same 
time that IFN-.sub..gamma. was administered. 
A first experiment assessed the effect of IFN-.sub..gamma. on Survival 
following suture challenge. Survival of an IFN-.sub..gamma. group (n=16) 
was compared to a control group (n=16). Surviving mice were observed for a 
minimum of 3 weeks after bacterial challenge to assess late mortality. 
A second experiment determined the effect of IFN-.sub..gamma. on local and 
systemic bacterial recovery following suture challenge. An 
IFN-.sub..gamma. group (n=26) was compared to a control group (n=35). Six 
to eight animals from each group were sacrificed by cervical dislocation 
at 1, 2 and 3 days after suture challenge. Blood was withdrawn immediately 
from each sacrificed animal by cardiac puncture. The medial compartment 
musculature, including the inserted suture, was excised "en bloc" and the 
suture separated and its length measured with a micrometer. Blood and 
muscle then underwent quantitative bacteriology. The number of mice that 
died during the experiment was recorded. 
To estimate the initial bacterial inoculum, the number of bacteria per 
millimeter of suture was determined by homogenizing a 1 cm length of the 
suture with 10 ml of sterile phosphate buffer saline (PBS), using a glass 
mortar and electrically-driven Teflon.RTM. pestle. After agitation for 5 
minutes, the homogenate was serially diluted in PBS, plated on nutrient 
agar (BBL Microbiology Systems, Cockeysville, Md.), and incubated 
overnight at 37.degree. C., after which the bacterial colonies were 
counted. The number of bacteria initially introduced into an animal was 
equal to the suture length in millimeters multiplied by the number of 
bacteria per millimeter in the suture. 
Quantitative determination of wound bacteriology was performed in a similar 
manner. Each excised specimen of muscle, together with the inoculating 
suture, was homogenized and plated as above. Values for bacterial quantity 
were determined as log.sub.10 of blood bacterial counts or log.sub.10 % 
local bacterial recovery from muscle. Of the blood removed by cardiac 
puncture, 0.1 ml was serially diluted with PBS and plated on nutrient 
agar. After overnight incubation, the number of colony-forming units was 
counted. 
The Student's t Test for independent means was used to analyze muscle and 
blood culture results, and the Chi Square test with Yates' correction for 
continuity was used in the analysis of survival data. In all cases, 
differences were considered significant at p&lt;0.05. 
The intramuscular dose of Klebsiella introduced with the suture ranged 
between 1.5 and 2.2.times.10.sup.6 organisms. The IFN-.sub..gamma. treated 
group had significantly greater survival than the control group from day 3 
onwards as is illustrated in FIG. 1. In FIG. 1 the survival of mice 
treated for 5 days prior to suture challenge is plotted using squares to 
indicate treatment with RPMI-1640 medium (controls) or using circles to 
indicate treatment with IFN-.sub..gamma.. There were no further deaths 
after day 6, and 10 mice in the IFN-.sub..gamma. treated group survived as 
compared to only 2 mice in the control group. 
In the second experiment, significantly more mice in the control group died 
(35 of 35) than in the IFN-.sub..gamma. group (4 of 26) as set forth in 
Table I. 
TABLE 1 
______________________________________ 
Number of Animals Sacrificed for 
Quantitative Cultures and Mortality 
At Each Time Period 
Mortality at Control Group 
IFN-.gamma. Treated 
Each Time Period 
(n = 35) Group (n = 26) 
______________________________________ 
Day 1 
Sacrificed 7 7 
Died 6 1 
Day 2 
Sacrificed 7 7 
Died 4 1 
Day 3 
Sacrificed 6 8 
Died 5 2 
______________________________________ 
The number of animals sacrificed for quantitative bateriology is also shown 
in Table 1. As illustrated in FIG. 2, the IFN-.sub..gamma. treated group 
had significantly less systemic blood-borne bacterial counts than the 
control group for each time period. In FIG. 2, the effect of 
administration of IFN-.sub..gamma. on blood cultures of mice treated for 5 
days prior to suture challenge is plotted using squares to indicate 
treatment with RPMI-1640 medium (controls) or using circles to indicate 
treatment with IFN-.sub..gamma.. 
There was no difference in local bacterial recovery at the site of 
infection between the IFN-.sub..gamma. treated group and control group for 
any time period. 
Pretreatment of mice with IFN-.sub..gamma. significantly reduced the 
mortality observed using the model. This indicates that IFN-.sub..gamma. 
treatment alters the curse of surgical wound infection. 
In the second experiment, recovery of bacteria at the local site of 
infection was not altered in IFN-.sub..gamma. treated mice; however, 
recovery of bacteria from the circulation of IFN-.sub..gamma. treated mice 
was reduced significantly compared to controls. This suggests that the 
IFN-.sub..gamma. treatment enhances the ability of the host to limit the 
spread of bacterial infections. The limitation on the spread of bacterial 
infection may contribute to the decreased mortality observed in mice 
treated with IFN-.sub..gamma.. 
EXAMPLE 2 
The efficacy of IFN-.sub..gamma. in prophylaxis and therapy was studied in 
a simulated wound infection model using a typical surgical pathogen. 
Groups of 12 CBA/J mice were subcutaneously injected with either murine 
IFN-.sub..gamma. Or RPMI-1640 medium (controls). The murine 
IFN-.sub..gamma. was produced by recombinant DNA technology (specific 
activity 2.3.times.10.sup.7 units/mg protein) and was a gift of Genentech, 
Inc. Bacterial challenge consisted of intramuscular injections of K. 
Pneumoniae (10.sup.3 organisms in 0.1 ml of physiological saline). 
Mice pretreated with IFN-.sub..gamma. at a dose of 7,500 or 750 units per 
day for 3 days, infected, and then treated therapeutically for 2 days 
survived significantly longer than controls or mice treated with 150 units 
of IFN-.sub..gamma. per day. Significantly (p&lt;0.05) greater survival than 
controls was observed with pretreatment for 5 or 3 days with 
IFN-.sub..gamma., but not with 1 day of pretreatment. 
Administration of IFN-.sub..gamma. to the opposite hind limb from the one 
receiving bacterial challenge was as effective as treatment in the same 
limb. When IFN-.sub..gamma. treatment was commenced 1 hour after bacterial 
challenge and continued for 7 days, 13 of 60 mice survived, a 
significantly (p&lt;0.05) greater number than the 4 of 60 surviving controls. 
These data confirm that IFN-.sub..gamma. is effective in pretreatment of 
Klebsiella pneumoniae infection of mice. Furthermore, these results 
support the therapeutic efficacy of IFN-.sub..gamma. in treatment of an 
extracellular bacterial infection associated with trauma. 
EXAMPLE 3 
Adult male CBA/J mice, each weighing 20-25 g (Jackson Laboratories, Bar 
Harbor, Maine), were anesthetized and were burned. Immediately after 
administration of a burn wound, bacteria were topically applied to the 
burn wound. The mice were anesthetized using 3.75 mg 
Ketamine=hydrochloride (Bristol Laboratories, Syracuse, N.Y.) and 0.5 mg 
Xylazine= (Miles Laboratories, Shaunee, Kansas) in 0.1 ml of physiological 
saline injected intramuscularly into the right hind limb. The dorsum was 
clipped with an animal clipper. 
The animals were then placed in a mold which was specially designed to 
expose a predetermined amount of the dorsum while protecting the remainder 
of the mouse from thermal injury. The mold consisted of a plastic cylinder 
lined with styrofoam with a large opening on one side through which the 
mouse was inserted and a smaller opening on the opposite side which 
exposed an area of the clipped dorsum equal to 30% of the total body 
surface of the mouse. Full thickness burns were consistently produced by 
immersion of the exposed dorsum in an 85.degree. C. water bath for 8 
seconds. The mice were resuscitated with 2 cc of Ringers lactate 
administered intraperitoneally. 
Two different bacterial challenges were used: K. pneumoniae (capsular type 
2) and P. aeruginosa. Immediately after resuscitation, bacteria (10.sup.8 
organisms) were administered topically to the burn wound in 1 ml of 10% 
gelatin using a sterile swab. 
Murine IFN-.sub..gamma. was administered subcutaneously into the left hind 
limb in daily doses of 7500 units in 0.1 ml injections. 
The murine IFN-.sub..gamma., as described in Example 1, was diluted with 
RPMI-1640 medium. Treatment commenced 5 days prior to burn and bacterial 
challenge, and the last dose was administered prior to this challenge. In 
all experiments, control animals received 0.1 ml of RPMI-1640 medium 
subcutaneously at the same time that the IFN-.sub..gamma. was administered 
to the experimental animals. 
Chi Square tests with Yates correction were used to determine the 
significance of differences in values obtained in the survival experiments 
set forth below. The Student's t Test was used to compare groups in 
experiments involving the expression of Ia antigens on mononuclear cells. 
In all cases, differences were considered significant at p&lt;0.05. 
Four experiments were performed. The purpose of the first experiment was to 
assess the effect of thermal injury alone on survival. Twelve untreated 
mice were burned and given no bacterial challenge. 
Burning without bacterial challenge produced no mortality. All twelve mice 
survived for three weeks following burning. 
The second experiment assessed the effect of IFN-.sub..gamma. on survival 
of mice with K. pneumoniae burn wound infection. Survival of an 
IFN-.sub..gamma. group (n=47) was compared to a control group (n=48). 
When mice had K. pneumoniae burn wound infection, there was significantly 
greater survival in the IFN-.sub..gamma. treated group than the control 
group from day 3 onwards, as illustrated in FIG. 3 in which a square 
denotes a value for the control group, a circle denotes a value for the 
experimental group and an asterisk denotes a value significantly different 
from the value for the control group on the indicated day. There were no 
deaths after day 8 of the experiment. Fifteen of 47 mice survived in the 
group treated with IFN-.sub..gamma. survived compared to 7 of 48 surviving 
mice in the control group. 
Burn alone significantly decreased mononuclear cell Ia antigen expression 
compared to controls on days 1 and 3, as illustrated in FIG. 4. In FIG. 4, 
square denotes a value for the control group, a triangle denotes a value 
for the burned but untreated group, a circle denotes a value for the 
experimental (burn plus IFN-.sub..gamma.) group, an asterisk denotes a 
value significantly different from the value for the control group on the 
indicated day, and the error bars indicate the standard error of the mean. 
The Ia antigen expression returned to normal by day 5 post-burn. Treatment 
of burned mice with IFN-.sub..gamma. prevented the drop in mononuclear 
cell Ia antigen expression on day 3 post-burn. In animals which were not 
burned, there was no difference between the control group and the group 
receiving IFN-.sub..gamma. alone (not shown) at any time. 
The third experiment assessed the effect of IFN-.sub..gamma. on survival of 
mice with P. aeruginosa burn wound infection. Survival in an 
IFN-.sub..gamma. group (n=27) was compared to survival in a control group 
(n=28). 
When mice had P. aeruginosa burn wound infection there was no difference 
observed in survival between the IFN-.sub..gamma. treated group and the 
control group. 
In the foregoing three experiments, surviving mice were followed for a 
minimum of 3 weeks after bacterial challenge to assess late mortality. 
The fourth experiment assessed the effect of burn and IFN-.sub..gamma. 
treatment on peripheral mononuclear cell Ia antigen expression. One 
hundred and forty one mice were divided into four groups. One group 
received only a burn (n=39), one group received a burn and 
IFN-.sub..gamma. (n=39), and a control group (n=39) received no treatment. 
The final group received only IFN-.sub..gamma. (n=24). Thirteen animals in 
each of the first three groups and 8 from the IFN-.sub..gamma. alone group 
were sacrificed on days 1, 3 and 5 post burn. Blood was obtained by 
cardiac puncture and mononuclear cells were assayed for levels of Ia 
antigen expression. 
Aliquots (100.mu.l ) of heparinized blood from each mouse were stained for 
20 minutes with a fluorescein isothiocyanate-conjugated monoclonal anti-Ia 
antibody (MA3 043, clone OX6, Accurate Chemical and Scientific Corp., 
Westbury, N.Y.). Erythrocytes were lysed with 1 ml of lysing reagent 
(Ortho Diagnostics, Raritan, N.J.). The specimens were then washed in 
buffer and fixed in 1% paraformaldehyde in phosphate buffered saline (pH 
7.4) prior to flow cytometric analyses. Stained samples were analyzed on 
an Ortho Cytofluorograph IIs flow cytometer (Ortho Diagnostics, Westwood, 
Mass.). Two thousand mononuclear cells were selected for fluorescent 
analysis by forward versus right angle light scattering properties. 
The percentage of cells staining for Ia antigen was recorded for each 
specimen. Complete white blood cell counts (obtained using a Coulter 
Counter, Coulter Electronics, Inc., Edison, N.J.) and Wright-stained 
microscope differential counts were made, and the absolute number of 
mononuclear cells was determined. The total Ia antigen expression per 
sample was calculated by multiplying the number of mononuclear cells by 
the value for the percent of cells staining positively for Ia antigen. 
When mice were treated for five days with IFN-.sub..gamma. prior to burn 
wound and bacterial challenge, survival was enhanced significantly in K. 
pneumoniae-infected mice compared to controls. No significant changes in 
survival were noted in mice which were pretreated with IFN-.sub..gamma., 
prior to a burn wound, for infection with P. aeruginosa. Different 
bacteria may respond to varying degrees to this treatment protocol in this 
burn wound infection model. 
Different protocols of IFN-.sub..gamma. treatment, perhaps combined with 
antibiotic chemotherapy, may be useful for successful therapy of P. 
aeruginosa infections. For example, in preliminary experiments, the 
addition of 2-5 days of daily subcutaneous injection of 7500 units of 
IFN-.sub..gamma. into the Ieft hind limb after burn and bacterial 
challenge appeared to increase resistance of burned mice to some strains 
of P. aeruginosa. Optimization of treatment protocols or of dosage of 
IFN-.sub..gamma., Or the combination of IFN-.sub..gamma. with other 
materials may contribute to further improved results. 
Because of its immunoregulatory properties, IFN-.sub..gamma., may play a 
crucial role in modulating host defenses against infection of burns. In 
the above experiments, decreased Ia antigen expression on mononuclear 
cells was observed in burned mice. Maintenance of Ia antigen expression in 
burned mice treated with IFN-.sub..gamma. may contribute to the enhanced 
resistance of mice to infection with K. pneumoniae. 
EXAMPLE 4 
Adult male CBA/J mice (H-2.sup.k) [Jackson Laboratories, Bar Harbor, Maine] 
weighing 20 to 25 g each were lightly anesthetized using ether and placed 
in a supine position. An approximately 2.5 cm midline abdominal incision 
was made, and care was taken to avoid injury to the abdominal contents. 
The abdominal cavity was lavaged with 3 ml of lactated Ringer's solution 
(Abbott Laboratories, Chicago, Ill.) and then closed using 4-0 nylon 
sutures. Next, the skin was closed with silk sutures, and normal saline 
(0.5 ml) was administered subcutaneously. Animals were then returned to 
their cages and were allowed food and water ad libitum. Mice that had 
undergone laparotomy ate and drank normally and did not show any signs of 
illness. Laparotomy without bacterial challenge produced no mortality. 
E. coli (10.sup.5 organisms) were administered in 1 ml intraperitoneal 
injections one-half hour prior to laparotomy. K. pneumoniae (capsular type 
2) was administered in 0.1 ml intramuscular injections into the right hind 
limb on day 5 following laparotomy, unless otherwise indicated. 
Murine IFN-.sub..gamma., as described in Example 1, was diluted with 
RPMI-1640 medium (GIBCO Laboratories, Grand Island, N.Y.). 
IFN-.sub..gamma. was administered in daily, tuboulaneous 0.1 ml injections 
of 7,500 units each. Therapy commenced 1 hour after laparotomy and the 
last dose was administered 1 hour prior to the second bacterial challenge. 
In all experiments, control animals received 0.1 ml of RPMI-1640 medium 
subcutaneously. 
A first experiment was directed at determination of an inoculum of E. coli 
that would cause an LD.sub.20 when injected intraperitoneally prior to 
laparotomy. Twenty-seven mice were divided into three equal groups. The 
groups received inocula of 10.sup.3, 10.sup.5, or 10.sup.8 organisms/ml 
and were observed for survival. 
Two further experiments assessed the effect of IFN-.sub..gamma. on survival 
in a dual bacterial challenge model. The first of the two experiments 
compared an IFN-.sub..gamma. -treated group with a control group (n=39 for 
both groups). Bacterial challenges were 10.sup.5 E. coli followed by 
10.sup.3 K. pneumoniae five days later. The second of the two experiments 
assessed the effect of reduction of IFN-.sub..gamma. therapy to 3 days 
(n=32 in both groups). Bacterial challenge was the same as in the previous 
experiment, except K. pneumoniae was administered 3 days after E. coli. 
An additional six experiments were carried out to determine whether 
IFN-.sub..gamma. used in combination with clinically relevant antibiotics 
in the laparotomy model caused any toxic effects. The antibiotic regimen 
and bacterial challenges used in each experiment are summarized in Table 
2. In each experiment, animals were divided equally into an 
IFN-.sub..gamma. treated group and a control group. 
TABLE 2 
__________________________________________________________________________ 
Number of Survivors in 
Animals in 
E. coli Klebsiella 
Each Group 
Antibiotics Used Each Group 
(Organisms/ml) 
(Organisms/ml) 
IFN-.gamma. 
Control 
__________________________________________________________________________ 
Cefazolin (1 mg/kg) + Tobramycin (1 mg/kg) 
24 10.sup.5 10.sup.4 
23 23 
Cefazolin (1 mg/kg) + Tobramycin (1 mg/kg) 
30 10.sup.8 10.sup.5 
4 3 
Cefazolin (1 mg/kg) + Tobramycin (1 mg/kg) 
28 10.sup.7 10.sup.5 
24 25 
Cefazolin (1 mg/kg) + Tobramycin (1 mg/kg) 
25 10.sup.8 10.sup.4 
6 6 
Sulfamethoxazole (50 mg/kg) + Trimethoprim (10 mg/kg) 
33 10.sup.8 10.sup.5 
27 26 
Sulfamethoxazole (25 mg/kg) + Trimethoprim (5 mg/kg) 
33 10.sup.8 10.sup.5 
25 23 
__________________________________________________________________________ 
In all of the above experiments, surviving mice were observed for a minimum 
of 3 weeks to assess any possible late mortality. 
Another experiment assessed the effect of laparotomy and IFN-.sub..gamma. 
on peripheral mononuclear Ia antigen expression. Two hundred and forty 
mice were divided into 4 equal groups. One group received only a 
laparotomy, one received a laparotomy and IFN-.sub..gamma., one received 
only IFN-.sub..gamma., and one received no treatment. Fifteen animals in 
each group were sacrificed on days 1, 3, 5 and 7 post-laparotomy (or 
following IFN-.sub..gamma. treatment in the control group). Blood was 
obtained by cardiac puncture and mononuclear cells assayed for levels of 
Ia antigen expression. 
Two different antibiotic regimens were used. In the first, cefazolin and 
tobramycin (Eli Lilly and Co., Indianapolis, Ind.) were used. Both drugs 
were administered in 0.1 ml intramuscular injections in the left hind limb 
at dosages of 1 mg/kg body weight. Cefazolin was administered 15 minutes 
after the first bacterial challenge, but prior to laparotomy. Cefazolin 
and tobramycin were then administered together daily commencing 1 hour 
after laparotomy and continuing for 5 days until the second bacterial 
challenge. Tobramycin was then administered alone daily for 3 days 
commencing 1 hour after the second bacterial challenge. 
In the second antibiotic treatment regimen, Bactrim.TM. (Roche 
Laboratories, Nutley, N.J.), a combination product of trimethoprim (16 
mg/ml) and sulfamethoxazole (80 mg/ml), was administered, after 
appropriate dilution, in 0.1 ml intramuscular injections in the left hind 
limb. The first injection was administered 15 minutes after first 
bacterial challenge. Bactrim.TM. was next administered 1 hour after 
laparotomy and daily thereafter until 3 days after the second bacterial 
challenge. Two experiments were performed using this regimen, a high dose 
experiment (10 mg/kg body weight trimethoprim) and a low dose experiment 
(5 mg/kg body weight trimethoprim). 
One hundred microliter aliquots of heparinized blood from each mouse were 
stained for 20 minutes with fluorescein-isothiocyanate-conjugated 
monoclonal anti-Ia (MAS 043p, clone 0X6, Accurate Chemical and Scientific 
Corp., Westbury, N.Y.). Erythrocytes were lysed with 1 ml of lysing 
reagent (Ortho Diagnostics, Raritan, N.J.). The specimens were then washed 
in buffer and fixed in 1% paraformaldehyde in phosphate buffered saline 
(pH 7.4) prior to flow cytometric analyses. 
Stained samples were analyzed on an Ortho Cytofluorograph IIs flow 
cytometer (Ortho Diagnostics, Westwood, Mass.). Two thousand mononuclear 
cells were selected for fluorescent analysis by forward versus right angle 
light scattering properties. A percent of cells staining for Ia antigen 
was recorded for each specimen. Complete white blood cell counts (Coulter 
Counter, Coulter Electronics, Inc., Edison, N.J.) and Wright-stained 
microscope differential counts were made, and an absolute number of 
mononuclear cells was determined. The total Ia antigen expression per 
sample was calculated by multiplying the number of mononuclear cells by 
the value for the percent of cells staining positively for Ia antigen. 
Chi Square tests were used to determine differences in the survival 
experiments. The Student's t Test was used to compare groups in the 
experiments involving the expression of Ia antigens on mononuclear cells. 
In all cases, differences were considered significant at p&lt;0.05. 
Mice infected with E. coli were assessed for survival 2 days after initial 
infection. Results are shown in FIG. 5. In FIG. 5, a square denotes values 
for the group which received 10.sup.3 organisms/ml, a triangle denotes 
values for the group inoculated with 10.sup.5 organisms/ml, and a circle 
denotes values for the group which received 10.sup.8 organisms/ml. No 
additional deaths occurred after that time. The dosage of 10.sup.5 
organisms/ml of E. coli was chosen as the first bacterial challenge for 
all additional experiments because this dose yielded the closest dose to 
an LD.sub.20. 
When mice received 5 days of therapy with IFN-.sub..gamma., there was 
greater survival in the IFN-.sub..gamma. treated group than in the control 
group, as illustrated in FIG. 6. In FIG. 6, a square denotes values for 
the control group, a circle denotes values for the IFN-.sub..gamma. 
-treated group, and an asterisk indicates a statistically significant 
difference between the values for the IFN-.sub..gamma. treated group and 
the control group on the date shown. When mice received only 3 days of 
therapy with IFN-.sub..gamma., there was also greater survival in the 
group treated with IFN-.sub..gamma. than in the control group, as 
illustrated in FIG. 7. In FIG. 7, a square denotes values for the control 
group, a circle denotes values for the IFN-.sub..gamma. -treated group, 
and an asterisk indicates a significant difference between the values for 
the IFN-.sub..gamma. -treated group and the control group on the indicated 
day. 
The number of mice surviving after day 15 is shown in Table 2. There were 
no further deaths after that time in any group. There were no significant 
differences in survival between IFN-.sub..gamma. treated and 
IFN-.sub..gamma. antibiotic-treated groups, as set forth in Table 2. This 
indicated no toxic effect of the dual modality therapy. However, this 
model was not designed to demonstrate a synergistic effect between 
IFN-.sub..gamma. and antibiotics. 
Laparotomy alone significantly decreased mononuclear cell Ia antigen 
expression compared to controls on days 1 and 3 post-surgery, as 
illustrated in FIG. 8. In FIG. 8, values for the control group are denoted 
by a square, values for the laparotomy alone group by a triangle, values 
for the laparotomy and IFN-.sub..gamma. treatment group by a circle, while 
an asterisk indicates a statistically significant difference. The Ia 
antigen expression returned to normal by day 5 post-surgery. Treatment of 
laparotomized mice with IFN-.sub..gamma. prevented the drop in mononuclear 
cell Ia antigen expression on days 1 and 3 post-surgery. 
EXAMPLE 5 
Eleven consecutive patients who sustained major trauma resulting in an 
Injury Severity Score ("ISS") [Baker et al., J. Trauma, 14, 187-196 
(1984)] of at least 20 were studied. These patients sustained the 
following injuries: burns (5 patients), gunshot wounds (3 patients), motor 
vehicle accidents (2 patients), and crush injury (1 patient). Blood 
samples were obtained within 5 days of injury. Ten healthy normal subjects 
were also studied. 
Three series of experiments were performed. The first assessed the effect 
of trauma on monocyte HLA-DR expression. An experiment assessed the effect 
of IFN-.sub..gamma. on monocyte HLA-DR antigen expression in mononuclear 
cell culture in trauma patients (n=11) and normal subjects (n=10). Four 
patients had serial tests performed throughout their hospital stay. The 
final experiment assessed the effect of IFN-.sub..gamma. on monocyte 
HLA-DR antigen expression in pure monocyte culture for trauma patients 
(n=5) and normal subjects (n=4). 
Human affinity-purified IFN-Y (Interferon Sciences, Inc., New Brunswick, 
N.J.) was used. The IFN-.sub..gamma. had a specific activity of 
approximately 1.0.times.10.sup.6 units/mg protein and was diluted with 
sterile phosphate-buffered saline. IFN-.sub..gamma. (500 units) were added 
to each culture. In all experiments, controls were set up with culture 
media alone added. 
Mononuclear cells were isolated and cultured as follows. Twenty milliliters 
of venous blood were collected in acid citrate dextrose anticoagulate 
tubes (Becton-Dickinson, Rutherford, N.J.). Mononuclear cells were 
separated from whole blood diluted 1:4 with RPMI-1640 medium (Gibco 
Laboratories, Grand Island, N.Y.) by centrifugation for 30 minutes at 1500 
rpm after layering on Ficoll hypaque-1077.TM. gradients. (Sigma Chemical 
Co., St. Louis, Miss.) at a 4:3 ratio. The mononuclear layer was 
harvested, washed twice with RPMI-1640 medium and resuspended in RPMI-1640 
medium supplemented with 5% human AB serum (Hazelton Laboratories, 
Hazelton, Pa.), 2.times.10.sup.-5 -mercaptoethanol, 300 units of 
penicillin, 300 mg of streptomycin and 75 pg of amphotericin B. 
Mononuclear cell cultures were established with 2.times.10.sup.6 cells in 
1.5 ml volumes per siliconized glass tube and incubated for 48 hour at 
37.degree. C. in 5% CO.sub.2. 
Monocytes were purified and cultured as follows. Twenty milliliters of 
venous blood were collected in EDTA anticoagulate tubes (Becton-Dickinson, 
Rutherford, N.J.). Whole blood was diluted 10:1 with dextran 500, and 
allowed to stand for 1 hour in room temperature to permit neutrophils to 
settle. Plasma was extracted, passed over Nycodenz.TM. (Accurate 
Chemicals, Westbury, N.Y., and then centrifuged for 20 minutes at 2000 
rpm. Purified monocytes were collected and washed with RPMI-1640 medium. 
These purified monocytes were then resuspended and cultures were 
established as previously described. 
The expression of HLA-DR antigen on monocytes was determined using dual 
monoclonal antibody staining and flow cytometry. One million cells of each 
preparation were stained with anti-human fluorescein coupled MO.sub.2 
monoclonal antibody (Coulter Immunology, Hialeah, Fla.), which 
specifically stains monocytes, and with isothiocyanate-coupled anti-human 
HLA-DR antigen (Becton-Dickinson, Sunnyvale, Calif.). Samples were 
analyzed on an Orthocytofluorograph IIs flow cytometer (Ortho Diagnostics, 
Westwood, Mass.) configured for simultaneous 2-color (red and green) 
fluorescent analysis. Gain settings were set on stained calf thymus 
standards (Fluortrol.TM., Ortho Instruments, Westwood, Mass.). In this 
analysis, HLA-DR antigen expression was measured only on cells which 
co-stained positively for MO.sub.2 antigen, thereby excluding all cell 
types other than monocytes from the analysis. 
In all experiments, two parameters were measured, the percentage of 
monocytes which expressed HLA-DR antigen and the density of the HLA-DR 
antigen expression on monocytes. The density of HLA-DR antigen expression 
on monocytes was measured as the mean fluorescent channel on gated 
positive cells (1000 channel resolution). 
The Student's t Test was used to determine differences. Differences were 
considered significant at p&lt;0.05. 
The mean HLA-DR antigen density on monocytes of trauma patients was 
85.8.+-.15.5 for normal subjects. The mean value for percentage of 
monocytes expressing HLA-DR antigen of trauma patients was 31.6% .+-.6.5 
and was significantly less than the mean of 91.1% .+-.2,5 for normal 
subjects. 
Mixed lymphocyte and monocyte culture alone, without IFN-.sub..gamma. 
addition, increased the density of HLA-DR antigen expression on monocytes 
in both normal subjects and trauma patients. IFN-.sub..gamma. treatment 
significantly increased the density of monocyte HLA-DR antigen expression 
above culture alone values in both normal subjects and trauma patients, 
although the levels for trauma patients treated with IFN-.sub..gamma. were 
less than the normal level, as illustrated in FIG. 9. In FIG. 9, an empty 
bar represents a baseline value, a hatched bar represents a value for 48 
hour culture, a solid bar represents a value for 48 hour cultures with 
IFN-.sub..gamma., and an error bar indicates the standard error of the 
mean for each value. 
IFN-.sub..gamma. treatment significantly increased the percentage of 
monocytes expressing HLA-DR antigen above culture alone and baseline 
values, as illustrated in FIG. 10. In FIG. 10, an empty bar represents a 
baseline value, a hatched bar represents a value for 48 hour culture, a 
solid bar represents a value for 48 hour culture with IFN-.sub..gamma., 
and an error bar indicates the standard error of the mean for each value. 
This value was not different from the baseline value for normal subjects 
and approached the IFN-.sub..gamma. value for normal subjects. 
In purified monocyte culture, culture alone significantly increased both 
density of monocyte HLA-DR antigen expression, as depicted in FIG. 11, and 
increased the percentage of monocytes expressing HLA-DR antigen above 
baseline values in the trauma group but not in the normal subjects group, 
as depicted in FIG. 12. In FIGS. 11 and 12 an empty bar represents a 
baseline values, a hatched bar represents values for 48 hour culture, a 
solid bar represents a value for 48 hour culture with IFN-.sub..gamma., 
and an error bars indicates the standard error of the mean for each value. 
IFN-.sub..gamma. treatment significantly increased the density of HLA-DR 
antigen expression in the trauma and normal groups. 
When IFN-.sub..gamma. treated cultures were considered, there was no 
difference between the trauma or normal groups in either density of HLA-DR 
antigen expression on monocytes, as shown in FIG. 11, or in the percentage 
of monocytes expressing HLA-DR antigen, as shown in FIG. 12. 
Of the four serially-followed patients, two made uneventful recoveries, and 
one developed major sepsis. IFN-.sub..gamma. treatment markedly increased 
monocyte HLA-DR antigen expression at each time. The other patient died of 
burn wound infection, and IFN-.sub..gamma. treatment increased monocyte 
HLA-DR antigen expression at each time, even for the final sample, which 
was obtained immediately prior to death. 
FIG. 13 is a graphic depiction of monocyte HLA-DR antigen values for the 
cultures obtained from the burn patient who died. In FIG. 13, baseline 
values are denoted by squares, 48 hour culture values by triangles, and 48 
hour culture with IFN-.sub..gamma. values by circles. 
The results confirm that severely injured patients have depressed levels of 
HLA-DR antigen expression as compared to controls. When IFN-.sub..gamma. 
was added to peripheral blood cell cultures obtained from these patients, 
the IFN-.sub..gamma. treatment resulted in an increase in HLA-DR antigen 
density of expression on monocytes. This increase in density of HLA-DR 
antigen expression did not result in a return to the normal range of 
HLA-DR antigen expression of controls, but there was a positive response 
to the IFN-.sub..gamma. treatment. 
When the percentage of monocytes expressing HLA-DR antigen was considered, 
a return to a near normal percentage of cells expressing HLA-DR antigen 
was observed in IFN-.sub..gamma. -treated peripheral blood cell cultures 
of trauma patients. Similar patterns were observed when cultures of 
purified monocytes were used, indicating that interaction with lymphocytes 
in culture may not play a role in this result. 
The kinetics of the response to IFN-.sub..gamma. were observed in the 
culture of the peripheral blood cells of a burn patient who succumbed to a 
septic infection. From these data, it is clear that HLA-DR antigen 
expression was depressed on the first day following injury, but that 
IFN-.sub..gamma. was capable of enhancing the percentage of monocytes 
expressing HLA-DR antigen until the last day of life for this patient. 
The results of this study indicate that HLA-DR antigen expression is 
depressed on monocytes of trauma patients, but that IFN-.sub..gamma. 
treatment may at least partially restore HLA-DR antigen expression. The 
resistance to infection of many trauma patients may be compromised as a 
result of impaired antigen presentation due to depressed HLA-DR antigen 
expression. In trauma patients, who are often unresponsive to conventional 
therapy, IFN-.sub..gamma. may be used to help restore immune defenses. 
EXAMPLE 6 
In an experiment generally following the procedures of Example 4, blood was 
collected from 10 normal subjects (control group) and 11 patients who 
sustained severe trauma (trauma group). A baseline MO.sub.2 DR value for 
each group was determined using dual monoclonal antibody staining and flow 
cytometry. Polk et al., Ann. Surg., 204, 282-299 (1986). The mononuclear 
layer of each blood sample was then harvested and incubated with either 
500 I.U. IFN-.sub..gamma. (IFN-.gamma.-culture) or control media (culture 
alone). The MO.sub.2 DR value for each sample was determined after 48 
hours of incubation. 
The mean baseline MO.sub.2 DR value for the trauma group was 32%, which was 
significantly less than the mean MO.sub.2 DR value of 91% for the control 
group (p&lt;0.001). In the trauma group, IFN-.sub..gamma. cultured MO.sub.2 
DR value was 81% which was significantly greater than both the mean 
baseline MO.sub.2 DR value of 32% (p&lt;0.001) and the 49% mean value of 
MO.sub.2 DR value when cultured without IFN-.sub..gamma. (p &lt;0.01). In the 
control group, there were no significant differences among the 
IFN-.sub..gamma. culture MO.sub.2 DR value (94%), the culture alone 
MO.sub.2 DR value (86%) or the baseline MO.sub.2 DR value (91%). These 
data confirm that HLA-DR antigen expression on monocytes of severely 
injured patients is markedly reduced and show that it may be increased 
significantly by IFN-.sub..gamma.. IFN-.sub..gamma. may be an important 
augmentor of host defense mechanisms in these patients. 
EXAMPLE 7 
Patients with severe injury [ISS &gt;20 and 1+or 2+bacterial contamination of 
their wounds, scored according to the procedure of Hershman et al., 
Injury, 19, 263-266 (1988)] received recombinant human IFN-.sub..gamma. 
(Genentech, Inc., South San Francisco, Calif.) at doses of 0.01 to 0.1 
mg/m.sup.2 /day for 7-10 days immediately following hospitalization. 
Standardized prophylactic antibiotic therapy with cefoxitin was 
administered during the first 72 hours. Patients were monitored for 
infection and monocyte DR antigen expression, the latter being an 
immunologic endpoint used as an indicator of clinical outcome. 
Treatment at all dose levels was extremely well tolerated. Fever was the 
only drug-related toxicity observed, and fever was only observed at the 
two higher doses. Among the first 13 patients treated, there were only two 
episodes of major sepsis and no deaths related to sepsis. Monocyte DR 
antigen expression, which is depressed in severe trauma victims, was 
restored to normal or greater than normal levels. Individual case 
summaries for five of the patients treated on this study are as follows. 
Patient BD was a 20 year old male who had suffered full thickness 
esophageal burns due to the ingestion of a caustic cleaning substance. His 
ISS was 25 and he had 1+bacterial contamination in his wound. He received 
IFN-.sub..gamma. therapy at a dose of 0.01 mg.m/.sup.2 for seven days and 
cefoxitin prophylaxis for 72 hours. He recovered fully from this injury 
without major sepsis. 
Patient TR was a 24 year old female who suffered a gunshot wound to the 
abdomen with injury to the pancreas and inferior vena cava. Her ISS was 25 
and she had 1+ bacterial contamination of her wound. She was treated with 
IFN-.sub..gamma. at a dose of 0.01 mg/m.sup.2 for seven days and cefoxitin 
prophylaxis. Nafcillin was also administered. Other than a culture of 
Staphylococcus aureus from her central venous catheter tip, the patient 
had no evidence of infection and recovered fully from her injury. 
Patient MD was a 28 year old male who suffered multiple stab wounds to the 
head and chest. His ISS was 26 with 1+bacterial contamination of the 
wound. The patient received recombinant IFN-.sub..gamma. therapy at a dose 
of 0.01 mg/m.sup.2 for seven days as well as cefoxitin antibiotic 
prophylaxis. The patient recovered fully and had no evidence of major 
sepsis. 
Patient BD was a 27 year old female who suffered severe pelvic injury due 
to a motor vehicle accident. The ISS was 25 with 1+ bacterial 
contamination of the wound. The patient was treated with IFN-.sub..gamma. 
at a dose of 0.01 mg/m.sup.2 for seven days as well as cefoxitin 
antibiotic prophylaxis. In addition, flagyl and amikacin were 
administered. The patient recovered fully from her injury without evidence 
of major sepsis. 
Patient BD was a 27 year old male who suffered a gunshot wound to the 
abdomen. His ISS was 25 with 1+ bacterial contamination. He received 
recombinant IFN-.sub..gamma. therapy at a dose of 0.1 mg/m.sup.2 for seven 
days as well as cefoxitin antibiotic prophylaxis. The patient developed an 
intra-abdominal abscess which required drainage and further antibiotic 
therapy, but then recovered fully from this injury. 
EXAMPLE 8 
Adult female Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, 
Ind.) weighing 190-230 g were subjected to hemorrhagic shock. The rats 
were anesthesized with intraperitoneal Ketamine (50 mg/kg) and 
Xylazine.TM. (15 mg/kg) and bled to a mean arterial pressure of 45 mm Hg 
which was maintained for 45 minutes. Animals were resuscitated with their 
shed blood and approximately 1.5 volumes normal saline. The mortality of 
this hemorrhagic shock protocol was approximately 15%; all deaths occurred 
within 24 hours following shock and these animals were excluded from 
analysis. 
Staphylococcus aureus 502A (ATCC 27172, American Type Culture Collection, 
Rockville, MA.) was maintained on trypticase soy agar at 4.degree. C. 
Prior to use, the bacteria were grown overnight in trypticase soy broth at 
37.degree. C., washed and resuspended in normal saline at a concentration 
of 4.times.10.sup.8 colony forming units (CFU) per ml. The bacterial 
concentration was determined by measuring the optical density at 620 nm 
and confirmed by serial dilution and backplating on trypticase soy agar. 
Recombinant rat IFN-.sub..gamma. was purchased from Amgen Biologicals 
(Torrence, Calif.) and had a specific activity of 4.8.times.10.sup.6 U/mg. 
IFN-.sub..gamma. was diluted with phosphate buffered saline containing 2% 
rat serum and was stored at -70.degree. C. prior to use. 
One hour after resuscitation from hemorrhagic shock, animals were injected 
with 5 separate inocula of 1.times.10.sup.8 CFU (0.25 ml) Staphylococcus 
aureus ("S. aureus") subcutaneously on the dorsum. Animals were divided 
into four treatment groups: a first group of control animals were 
resuscitated following shock but received no further treatment; a second 
group of antibiotic-treated animals received cefazolin ("CEF") (Smith, 
Kline and French, Philadelphia, Pa.) intraperitoneally at 30 mg/kg, 30 
minutes prior to inoculation and then again 4 hours later; a third group 
of IFN treatment-animals received IFN-.sub..gamma. 7500 U subcutaneously, 
one hour following inoculation and then daily for three days; and a fourth 
group received both CEF and IFN-.sub..gamma. as in groups 2 and 3. Tissue 
antibiotic levels were measured in the subcutaneous tissue 30 minutes 
after the initial dose of CEF by a standard bioassay. 
Animals were sacrificed on day 7 by cervical dislocation under anesthesia. 
A dorsal incision was made and the skin reflected. The diameter of each 
abcess was measured in situ with a micrometer, and abcesses were dissected 
free from the musculature of the back. Abscesses were then excised from 
the skin and weighed. 
Statistical analysis of abscess number was done using Fisher's Exact Test. 
Comparisons of abscess diameter and weight were made using a prior 
analysis of variance followed by a Tukey - Kramer HSD Test. Significance 
was set a priori at p &lt;0.05. 
The results for abscess number, diameter and weight are summarized in Table 
3. In Table 3, for values indicated by the ".sctn." symbol, a significant 
difference from the control at the p&lt;0.05 vs. level was determined, and 
for values identified by an asterisk, a significant difference from the 
value for CEF alone was determined at the p&lt;0.05 level. 
TABLE 3 
______________________________________ 
EXPERI- 
MENTAL ABSCESS ABSCESS ABSCESS 
GROUPS #(n = 20) DIAMETER (mm) WT (mg) 
______________________________________ 
Control 20 13.4 .+-. 1.9 504 .+-. 125 
CEF 20 .sup. 10.1 .+-. 1.1.sup..sctn. 
191 .+-. 78.sup..sctn. 
IFN 20 14.6 .+-. 3.8 769 .+-. 212 
CEF + IFN 14* 4.8 .+-. 1.6* 
.sup. 67 .+-. 59.sup..sctn. 
______________________________________ 
The administration of standard antibiotic prophylaxis (CEF) following 
hemorrhagic shock decreased abscess diameter and weight compared to 
control but did not decrease abscess number. Animals treated with 
IFN-.sub..gamma. alone had abcesses which were larger than those of 
control animals. This correlated with a subjective impression of an 
increased inflammatory reaction around these lesions. The addition of 
IFN-.sub..gamma. to CEF significantly decreased abscess number, diameter 
and weight as compared to the control or to the use of CEF alone. Analysis 
of the 99% confidence intervals of the mean abscess diameter for each 
group illustrates the synergy between these immune adjuvants and CEF in 
decreasing abscess diameter as compared to controls or, as illustrated in 
FIG. 14, as compared to either modality alone. 
Tissue CEF levels were 14.9.+-.5.2 .mu.g/gm 30 minutes after the initial 
dose, exceeding the minimal inhibitory concentration employed for the S. 
aureus (0.15 .mu.g/ml). 
Although the present invention has been described in terms of particular 
embodiments, it is understood that modifications and variations will occur 
to those skilled in the art. For example, when IFN-.sub..gamma. is 
administered via the intravenous route, antibiotics and IFN-.sub..gamma. 
may be co-administered. Therapy according to the present invention could 
be used for all types of trauma, and may be used for emergency or elective 
surgery. Classes of antibiotics useful according to the present invention 
include, but are not limited to, cephalosporins, penicillins (including 
semi-synthetic penicillins), bacteriostatic antibiotics and 
aminoglycosides. 
Accordingly, it is intended that the present invention include all 
variations and modification which come within the scope of the claims.