PB 230 933
INTERACTIONS OF VARIOUS AIR POLLUTANTS ON CAUSATION OF
PULMONARY DISEASE
James D. Fenters, et al
IIT Research Institute
Prepared fur:
Environmental Protection Agency
September 1973
DISTRIBUTED BY:
National Technical Information Service
U. S. DEPARTMENT OF COMMERCE
5285 Port Royal Road, Springfield Va. 22151
This document bas been approved for public release and sale.
-------
BIBLIOGRAPHIC DATA
HEET
Title anTSubiitle
1- Re-poii No.
EPA-650/1-73-002
PB 230 933
Interactions of Various Air Pollutants on Causation
of Pulmonary Disease
James D. Renters and Robert Z. Maigetter
S- Performing Organization Repi.
N7o.
Performing Organization Name and Address
I IT Research Institute
10 West 35th Street
Chicago, Illinois 60616
2. Sponsoring Organization Name and Address
! Environmental Protection Agency
Research Triangle Park
North Carolina 27711
15. Supplementary Notes
. Report Date
September T973
10. Project-Task'Hork Unii No.
11. Contract/Gram No.
68-02-0666
13. Type of Report & Period
Covered
Final
14.
This report consists of four studies. The materials, methods, and experiments
protocol tor each study were presented. The parameters of interest were hemagglutinat-
ion-inhibltion (HI) and serum neutralization (SN) antibody formation, serum immunoglo-
bulin levels, lung histopathology, mortality rates, lung lesion scores, and extent of
lung edema in mice. The four studies were: (1) immune response in mice during long
term exposure to nitrogen dioxide, C2> immune response in mice during a four-week ex-
posure to N0.2, \S) effect of chronic exposure to NOy on resistance to Klebsiella
pneumonlae, and (4> effects of manganese on resistance to respiratory infection. In
"the first study prior'to vaccination the mice were exposed continously to 2ppm N02,
O.Sppm NOo with 1-hr daily peaks of 2ppm H02 5 days in a week, or filtered air for 3
months. They were thereafter, held in either an N0£ environment or filtered air. At
specified time intervals, groups of mice were challenged by the respiratory route with
live influenza virus. The same N02 levels were utilized in the second and third study.
The N02 exposure time was different for each study. The morality rates and results of
measured parameters are evaluated and conclusions are presented for each study.
PRICES SUBJECT TO CHA.S.,
17. Key Vords and Document Analysis. 17a. Descriptors
Air pollution
Mice
Nitrogen dioxide
Manganese
Bacteria
Influenza virus
Immunization
Histology
Serology
Pneumonia
17b. Identifiers/Open-Ended Terms
Immunoglobulin^
Mortality rates
Hemagglutination-inhibition antibody
Serum neutralization antibody "'^ATIONAL TECHNICAL
Lung lesion INFORMATION SERVICE
U s Department ol Conmnrc.
1 in «v Springfield VA 22151
I7c- COSATI Field/Group 13B, 6F «" v
IB. Availability Statement
19. Security Class (This
Report)
UNCLASSIFIED
121. No. of Pages
'
20. Security Class (This
'J'NCI-ASSIFIED
FOREWORD
This is Report No. IITRI-L6069-4 (Final Report), entitled,
"Interaction of Various Air Pollutants on Causation of Pulmonary
Disease", Contract No. 68-02-0666, IITRI Project L6069. The
studies were conducted by IIT Research Institute for the
Environmental Protection Agency during the period from August
18, 1972 through August 17, 1973.
The principal investigator is Dr. Richard Ehrlich, the co-
investigator is Dr. James D. Fenters and the principal professional
associate is Dr. Robert Z. Maigetter. Other personnel partici-
pating in the program are Dr. C. D. Port, Mr. J. C. Findlay, Mr.
T. Sharp, Ms. E. Silverstein and Mr. S. Britton.
The experimental data are recorded in IITRI Logbooks C20951,
C2;056, C21281, C11790, C15809, C21474, C21475, D1756, D1812
and D1823'."
Respectfully submitted,
IIT RESEARCH INSTITUTE
AWBERC LIBRARY
U.S. EPA
26 'W. MARTIN LUTHER KING DR.
CINCINNATI, OHIO .45268
Approved by:
Robert Z. Maigetter
Research Microbiologist
Life Sciences Research
James D. Fenters v
Head, Microbiology Research
Life Sciences Research
Richard Ehrlich
Director
Life Sciences Research
RZM/JDF/sf
THIS FORM MAY BE REPRODUCED
USCOMM-DC l4BBZ->~/c
III RESEARCH INSTITUTE
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TABLE OF CONTENTS
II.
IMMUNE RESPONSE IN MICE DURING LONG-TERM
EXPOSURE TO NITROGEN DIOXIDE
A. Introduction
B. Materials and Methods
C. Results
1. Body Weights
2. Serology
3. Immunoglobulins
4. Infectious Challenge
a. Four Weeks after Vaccination
b. Sixteen Weeks after Vaccination
c. Twenty-eight Weeks after Vaccination
5. Lung Edema
6. Histopathology
D. Summary
IMMUNE RESPONSE IN MICE DURING A 4-WEEK EXPOSURE
TO N02
A. Introduction
B. Materials and Methods
C. Results
EFFECT OF CHRONIC EXPOSURE TO N02 ON RESISTANCE
TO K. PNEUMONIAE
A. Introduction
B. Materials and Methods
C. Results
IV. EFFECTS OF MANGANESE ON RESISTANCE TO
RESPIRATORY INFECTION
A. Introduction
B. Materials and Methods
C. Results
III RESEARCH INSTITUTE
ii
I. IMMUNE RESPONSE IN MICE DURING LONG-TERM EXPOSURE TO
NITROGEN DIOXIDE
III.
1-1
1-1
1-7
1-7
1-7
1-9
1-16
1-17
1-20
1-20
1-21
1-21
1-27
II-l
II-l
II-l
II-5
III-l
III-l
III-l
III-3
IV-1
IV-1
IV-1
IV-4
A.
Introduction
Previous studies conducted in our laboratories indicated
that chronic exposure to 1 or 5 ppm of nitrogen dioxide (N02)
markedly affected the ability of squirrel monkeys to produce seru
neutralization antibodies (Fenters, ejt al_. Am. Rev. Resp. Dis.
104, 448, 1971; Fenters, et al. Arch. Environ. Health 2_7, 85, 197
To elucidate the effect of chronic exposure to low concentrations
of N02 on the immunological response, further studies were con-
ducted in mice vaccinated with a highly purified influenza virus
vaccine. Parameters of interest were hemagglutination-inhibit ion
(HI) and serum neutralization (SN) antibody formation, serum
immunoglobulin levels, lung histopathology, and mortality rates,
lung lesion scores, and extent of lung edema in mice challenged
with live infectious influenza virus.
B.
Materials and Methods
Animals. Four-week-old specific-pathogen-free male Swiss
albino mice, CD-I strain, were obtained from Charles River Lab-
oratories. After a two-week quarantine period, the mice were
placed in the environmental chambers and held for two days before
initiation of the exposures. During the exposures, the mice were
removed from the chamber for one hour three times a week for
maintenance. Clean cages were provided once a week and food and
water were provided ad_ libitum.
Influenza Virus. Mouse-adapted influenza A2/Taiwan/I/64
virus was passaged several times in mice and 207. lung suspension
of the virus was used for all infectious challenges. Prior to
use, the virus was identified by use of specific antiserum ob-
tained from the National Institutes of Health.
IIT RESEARCH INSTITUTE
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Vaccine, Chick embryo A0/Taiwan/I/64 influenza vaccine
(Zonomune), Lot No. BP0549, was supplied by Eli Lilly and Company,
Indianapolis. Mice were vaccinated by a single subcutaneous
injection of approximately 279 CCA units in 0.1 ml vaccine.
HI and SN Titrations. Tests were performed in duplicate,
by the microtiter method in disposable V-plates (Cooke Engineering
Co., Alexandria, Va.) described by Davenport and Minuse (Diagnostic
procedures for Viral and Rickettsial Diseases, 3rd ed. Am. Public
Health Assoc., New York, 1964). In all tests, 1% chicken red
blood cells were used.
Four hemagglutinating units of antigen were used for the
HI test. All antisera were heat-inactivated at 56°C for 30 rain
and treated with trypsin-periodate to remove nonspecific inhibitors
of hemagglutination.
The protocol Osed fbr"ttfS test'was -similar -to- that * described
in the USPHS Requirements (Division of Biologies Standards, 6th
rev., 1947). Sera were heat-inactivated at 56°C for 30 min,
serially diluted, and incubated with an equal volume of influenza
virus for 1 hr at 4°C. The serum-virus mixture was then tested
in 10-day-old embryonated chicken eggs. The eggs were inoculated
by the allantoic route with 0.1 ml of the virus-serum mixture,
incubated at 37°C and harvested when an EID^Q (50% egg infectious
dose) dose of 32 to 320 was attained as indicated by hemagglutination
of the virus control. The £1050 dose was determined by parallel
infectivity tests in eggs by using a 0.1-ml virus-saline mixture.
Phosphate buffered saline (PBS), PBS plus normal mouse serum,
and normal mouse serum plus virus were inoculated into eggs as
controls.
Immunoglobulin Concentration. Quantitative radial immuno-
diffusion plates for mouse immunoglobulins IgA, IgG^, IgG2, and
IgM were procured from Meloy Laboratories, Inc., Springfield,
Virginia. Reference standards obtained from pooled sera of normal
mice were assayed daily, in duplicate, to provide quality control.
MT RESEARCH INSTITUTE
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Duplicate serum samples were placed in preformed wells, the plates
incubated at 22°C for 18 hr and the radial diffusion diameters
measured using a Bausch & Lomb 7x lens. Duplicate radial diffusion
diameters were recorded for each of the pooled serum sample; con-
sisting of seven to 10 pooled samples at each experimental point.
Nitrogen Dioxide Exposure. To maintain the control and
experimental mice under similar conditions, three identical
aluminum-lined chambers (4 x 6 x 6.5 ft) were used for the ex-
posures. Randomly selected mice were housed in suspended wire
cages, which were periodically rotated to various positions on
the cage racks. This assured a thorough and unbiased exposure
to the experimental environment. A minute amount of N02 was
continuously passed from & cylinder through a stainless steel
tube into a glass mixing vessel where it was diluted and mixed
with charcoal-filtered ambient air. The mixture was then passed
into the NC>2 exposure chambers at a rate of 20 changes per hour.
The same air flow pattern was maintained in the control chamber
where the chaircoal-filtered ambient air was used. To verify
the homogeneity of N02, air samples were taken from different
sections of the chambers and the N02 concentration determined
and calculated by the Saltzman method, A Mast NO-> gas analyzer
was used for continuous monitoring. The mean temperature in the
chambers was 24° + 1°C,
Scoring Pulmonary Lesions. The extent of pulmonary lesions
was expressed as the percentage of the total lung consolidated.
A score of 1 represented 25% lung consolidation, 2 = 50%, 3 = 75%,
4 = 100%, and a score of 5 represented animals that died during
the experiment.
Lung Edema. The ratio of the wet-to-dry lung weight of
lungs was used to express the extent of edema. Three pools of
three to five lungs each were used for each experimental group.
The pooled lungs were weighed immediately after removal from the
mice then dried in a vacuum desiccator and reweighed at 24-hr
intervals until there was no additional weight loss.
IIT RESEARCH INSTITUTE
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Aerosol Challenge. Infectious respiratory challenge was
conducted in a 350-liter plastic aerosol chamber (60 x 60 x 95 cm)
installed within a microbiological safety cabinet. A University
of Chicago Toxicity Laboratory type atomizer was used to produce
a viral aerosol with a majority of <5^.-mass-median-diameter airborne
particles. The microbial suspensions were fed to the atomizer
by a 50-ral syringe activated by a motor-driven piston delivering
the suspension at a rate of 0.4 ml/min. Filtered air was supplied
to primary and secondary inlets of the atomizer at a flow rate
of 33 liters/min. The chamber was maintained at 78 + 6% RH and
24° + 1°C.
The aerosol was sampled with an all-glass impinger (AGI-30)
containing PBS with 0.2% bovine serum albumin as a collecting
fluid. The inhaled dose was estimated on the basis of the con-
centration of the virus per liter of air, respiratory minute
volume of the mice, and the duration of exposure to the aerosol.
For the infectious challenge, groups of mice were placed in
the chamber and exposed to the aerosol for 5 to 6 min. After the
challenge, the mice were air-washed for 10 min, removed from the
aerosol chamber and held for 14 days in filter-capped cages in
a clean-air, isolated animal room.
Histopathology. The mice were anesthetized with carbon
dioxide, the lungs and heart removed as a unit, and representative
tissues were fixed in a 107.. phosphate buffered formalin solution.
After blocking in paraffin, 4u.-tb.ick sections were cut in a rotary
microtome, stained with hematoxylin and eosin, and examined.
Experimental Protocol. Prior to vaccination the mice were
exposed continuously for 3 months to one of the following three
environmental conditions:
2.0 ppm of N02 (2 ppm N02)
0.5 ppm of NCU with daily 1-hr peaks of 2.0 ppm of
N02 for 5 days /week (0.5/2 ppm
• filtered air (0 ppm NC>2)
After the 3-month exposure, the mice were vaccinated with influenza
vaccine by a single subcutaneous injection in the dorsal thoracic
area and thereafter held in either an N(>2 environment or filtered
air. Groups of 14 to 20 mice were killed at 2,4,8,12,16,20,24,
and 28 weeks after vaccination. Sera from two mice were pooled
and assayed for HI and SN antibodies and for immunoglobulin levels.
To determine the protective effect of the vaccine, as measured by
mortality rates and lung lesion scores, groups of 20 mice were
challenged with A2/Taiwan influenza virus at 4, 16, and 28 weeks
after the vaccination. Table 1-1 outlines the protocol for the
entire study.
Statistical Analysis. As appropriate, the experimental
results were subjected to statistical analysis and the significance
of the observed differences was reported at the <57o probability
level. The immunoglobulin level data were analyzed by a two-way
analysis of variance and a multiple range test was used to determine
the ranking and significance of the observed differences . The
significance of the differences in mortality rates was determined
by the normal approximation with correction for continuity (K. S.
Brownlee, Statistical Theory and Methodology in Science and
Engineering, p. 121, 1960, John Wiley and Sons, New York). This
method is equivalent to the chi-square test applied with a 2 x 2
contingency table, except for the use of corrections for continuity.
The Student t-test was used for statistical analysis of the lung
lesion scores.
IIT RESEARCH INSTITUTE
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IIT RESEARCH INSTITUTE
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Table 1-1
PROTOCOL FOR 1MMUNOLOGICAL STUDIES IN MICE
EXPOSED TO NITROGEN DIOXIDE
C.
Results
Pre-Vacci nation
N02 Exposure
A. 2 ppm
B . 0.5 ppm with
daily 1 hr
peaks of
2 ppm
C. 0 ppm
Vaccination
Treatment
(1) Vaccine
(n=440)
(2) Saline
(n=120)
Post -Vaccination
for 2,4,8,12,16
(la) 2 ppm
Ab & Ag at
(n-160)
(Ib) 0 ppm
Ab & Ig at
(n=160)
(2a) 2 ppm
(n-60)
(2b) 0 ppm
(n-60)
N02 Exposure
,20,24,28 wks
each period
each period
Identical experimental groups and obser
as the above but exposed to 0.5 ppm N02
daily 1 hr peaks of 2.0 ppm N02
(1) Vaccine
(n-660)
(2) Saline
(la) 0 ppm
Ab & Ig at
(n=160)
(Ib) 0.5/2 ppm
Ab & Ig at
(n=160)
(Ic) 2 ppm
Ab 6. Ig at
(n=160)
(2a) 0 ppm
(n=60)
(2b) 0.5/2 ppm
(n-60)
(2c) 2 ppm
(n=60)
each period
each period
each period
Infectious Challenge
at 4.16.28 wks
At each period
(n=60)
At each period
(n-60)
At each period
(n-60)
At each period
(n=60)
vations
with
At each period
(n=60)
At each period
(n=60)
At each period
(n-60)
At each period
(n-60)
At each period
(n=60)
At each period
(n-60)
Sera for initial HI and SN antibody titers were obtained from groups of 20 mice
after the 3 months pre-exposure to each experimental condition.
Ab and IG Studies: HI and SN antibody and immunoglobulin assays on pooled serum
samples consisting of 7-10 groups of 2 mice.
Mortality Studies: Infectious challenge with airborne A2/Taiwan influenza virus
of 3 groups of 20 mice.
1. Body Weights
Mice from two randomly selected cages in each of the three
exposure groups were weighed at weekly intervals. The initial
mean body weights of 22 mice in 2.0 ppm N02 chamber, 21 mice in
0.5/2 ppm NC>2 chamber, and 30 control mice were 30.0, 30.0, and
32.5 g. At the time of vaccination, i.e. 12 weeks after initiation
of the exposures, mice exposed to NC>2 had gained an average of
13 g while the control mice gained 9.5 g. All three groups of
mice weighed 44.5 g at 21 weeks after initiation of the exposure
and after 28 weeks the mean weights were 44.2, 45.9 and 45.1 g
for the 2.0 ppm NC>2 and 0.5/2 ppm NC>2, and control mice, respect-
ively. The respective mean weights at 40 weeks were 45.4, 46.9
and 46.3 g. Thus, all groups gained weight consistent through-
out the study.
2.
Serology
The differences in HI antibody levels between experimental
and control mice observed throughout the study were not significant
(fourfold) and the HI antibody titers appeared to decline at the
same rate in all animals (Table 1-2). Some differences were noted
in the levels of SN antibody. Two weeks after vaccination the
SN antibody titers appeared depressed in mice exposed to NC>2
(Table 2). A significant fourfold difference in SN antibody titers
was seen between control mice (1:34) and mice continuously exposed
to 0.5/2 ppm of NC>2, and those held in filtered air before
vaccination and 2 or 0.5/2 ppm of N02 after vaccination (<1:8 to
1:8). At the 4- and 8-week periods, the SN titers were comparable
for ;all groups. The decline in SN titers in all groups began
approximately 12 weeks after vaccination and appeared unaffected
by the experimental treatment.
1-6
IIT RESEARCH INSTITUTE
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Table 1-2
MEAN HI AND SN RECIPROCAL TITERS IN VACCINATED MICE EXPOSED TO NITROGEN DIOXIDE3
N02,ppm
3 Months Post-
0 0
0.5/2
2
0
0
0.5/2
2
0.5/2
2
0.5/2
2
0
0
18
13
17
33
12
9
6
HI Titerb
Weeks after
— 7 vF T-* —
16
16
11
16
8
19
23
14
10
7
11
8
16
12
L£.
14
12
7
1 30
14
14
IS
Vaccination
- 16
20
23
19
33
13
13
14
zu
7
14
14
14
10
7
16
m
<8
<8
<8
<8
<8
<8
<8
SN Titer
28
9
<8
<8
c
8
<8
<8
34
<8
12
8
8
10
20
Weeks after
23
31
46
26
26
15
40
B_
23
29
26
19
36
21
21
i£
10
7
14
15
11
8
17
Vaccination
21
17
12
21
8
25
17
20
7
8
7
10
6
12
24
24
10
9
11
8
17
17
16
17
9
8
c
12
16
11
Each titer represents a mean of seven to ten pools of serum, with two mice used per pool.
Four antigen units of egg-grown A2/Taiwan influenza virus were used.
, o ,,- -,1 week serum samples tested against 258-30 EID50 units
4,'8,24,28. week serum samples tested against 21-47 EID50 units
12 and 16 week serum samples, tested against 100-210 EID5Q units
20 week serum samples tested against 47-320 EID5Q units
Anim.tls not available for bleeding.
-------
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elative
Arrows indicate either a signi
increase -or decrease in the se
immunoglobulin concentration r
to filtered-air values.
c
•H
C
— 1
i— I
3
J2
O
3f immunogl
1-5 shows the concentrations i
, TD a c o x
tOOJ .COQJ E COQJ 4J
O co I O o 3 E E
•H -Oa4-i.H4-tcocO
• AJCO ^43ajcoM-icijtijo
WCOcOCOCU4-ll-ICU.rl^S^-t
O 4-1 .ft *H J»( CO CO 00 CO •• *rl
-HCcjr-fca cflcu^E^C
UO)33U -SoOmOlOO
QJCJBJ3 CO B ^-IM^-I
CXEcoO'DCOUcO'O4-' CO
OCOrJE-rlOnBtOU
COOEOOCfl^34J| CO^ii^tl
3 -HO Oat jj-DQ)
OB BijajcOr-ioOBco'W
-rl-rl -3OOOl*-la.BclJE-ll'-l
UM4JSUCO ^-I^CJ -H
c03Mgo aJW^BE-O
>J33.rICUOJ OOMCO-rl
OO oi-BcogcOO 4J
V41-I.HCO 4-> SP CO
OOO>)-I3.» cu C3C
X-lOU-lor^iD^cOcD^-lo
C-H'HVOWW .CrHJ^T)
CN3T3^lC^CO «4J3co-rl
OB COrHEXU J3 T3
3BCO> -rl-QCUJiOM
-riCO "SlJ-UCOr-lrfU
O SOJO^ltjlW-rlOO O.
4JB J3CNr-4Q>COr-lO -H
OTJ4J-*OcncoE CUM r-l
cucusiUE'-iSOCtJBtdCS
CJCJCOM3C^?.rl4J 4->OjO
•HBO |M 4JCOCCBO
B 0) p, CU B CUtO-DT-ICUaM
3X-BM-O-rl EOOO
73 M CU 4-> r-tBrldJCO-rlOO
OJ«-I •OsScO.CCUti B
4-IC4iiCO'-)in.rl>4JO(UX;3
COT-ICU4-1 Mn EQ.4-JB
B CUO- COM-IOCUX^HB
•rlOJ30JM|"^O4Jl-|CU3~-i
CJ -d 1 4-1 C0| O| OJ
o u oo IM MiHaJTi«-icocoj3
CO CNtO*J|r-(|ooa)l4-l3BCJ
>00 0)| 14-rli-IOOcO
EOJr^ •C03MT3-rl^-IOJ
IM ^H r! i— | n r— | >r_| Q p, ^ ^j
Oco4-*'ucootaco(XcucocoE
co OJBBX coj3>Mi-l
COOJtl^ico3MCO 4J U Si
^CQOJl-lOBcO CO copqSBU]rrji4-ij::aJv-i
CQ-
3 Months
Pre-Vacc
0
0.5/2
2
0
0
0.5/2
2
ppm
Post-
Vacc.
0
0.5/2
2
0.5/2
2
0
0
HI
~2
71
60
70
100
50
20
29
• $" 8
75
67
25
71
30
60
86
71
29
29
67
33
67
44
Response ,
%
~T2 T5 213 Z5 2~tT
57
57
14
83
43
56
44
71
86
57
67
57
29
43
29
-:43
'57
'60
/_
;57
• 14
57
0
0
0
0
0
14
14
25
0
0
b
20
14
16
~z ;
100
0
50
43
40
56
57
SN Response 7.
—zr — nr~
100
100
88
86
80
80
86
100
71
86
67
67
63
78
~nr~
57
43
33
67
43
44
56
TF~
57
67
71
83
43
43
43
IF"
33
43
43
60
29
57
57
24 "
43
67
33
40
71
57
100
~H
62
50
67
b
40
57
50
fl Each point represents a mean of seven to ten pools of serum, with two mice used per pool.
b Animals not available for bleeding.
-------
Table 1-5
SERUM IMMUNOGLOBULIN CONCENTRATIONS IN VACCINATED MICE
EXPOSED TO VARIOUS NITROGEN DIOXIDE CONDITIONS
NO^
, ppm
3 Months Post-
Pre-Vacc. Vacc.
0
0.5/2
2
0
0
0.5/2
2
0
0.5/2
2
0
0.
0.5/2
2
0
0.5/2
2
0
0
0.5/2
2
0
0.5/2
2
0
0
0.5/2
2
0
0.5/2
2
0
0
0.5/2
2
0
0.5/2
2
0.5/2
2
0
0
0
0.5/2
2
0.5/2
2
0
0
0
0.5/2
2
0.5/2
2
0
0
0
0.5/2
2
0.5/2
2
0
0
0
0.5/2
2
0.5/2
2
0
0
Weeks Immunoslobulin, rag/ml
Post-Vacc. l£A
2 0.700
0.620
0.435*
0.5001
0.535
0.600
0.550
4 0.515
0.560
0.700
0.770t
0.740
0.380
0.460
8 0.830
0.740
0.5101
1.200t
0.630
0.800
0.6501
12 1 . 046
0.824
1.052
1.117
1.297
0.840,
0.543*
16 0.973
1 . 088.
1.5787
1 . 88lt
1.909t
0.856
1.297
IgM
0.200
0.235
0.260
0.1151
0.190
0.250
0.260
0.180
0.245t
0.220
0.190
0.260
0.200
0.180
0.215
0.235
0.1301
0.175
0.210
0.225
0.220
0.182
0.211
0.196
0.246
0.259t
0.166
0.200
0.160
0.254
0.231
0.244
0.232
0.213
0.235
XsGl
1.75
5. SOT
3.80
3.95
5.60T
2.15
2.70
3.75
5.40
4.40
5.85t
6.80t
3.75
5.00
5.00
7.40T
6.20
5.60
4.50
6.30
6.20
4.80
9.20t
5.37
6.20
6.30
4.28
7.20
7.50
5.53
6.60
6.75
8.10
8.60
8.25
T p-QO
4.30
5.20
4.35
4.30
5.50f
4.10
3.80
4.05
6.50T
5.10
6.55'
6.301
4.75
5.22
5.60
6.85
5.22
6.30
5.85
6.00
6.75
4.50
5.59,
6.401
6.19t
7.84J
4.45
5.38
5.29
6.08
7.61f
6.22
7.35t
6.78
7.35
IIT RESEARCH INSTITUTE
1-12
Table 1-5 (continued)
N02, ppm
3 Months
Pre-Vacc.
0
0.5/2
2
0
0.5/2
2
0
0.5/2
2
0
0
0.5/2
2
0
0.5/2
2
0
0
0.5/2
2
Post- Weeks
Vacn. Post-Vacc.
0 20
0.5/2
9
0.5/2
7
£.
0
0
0 24
0.5/2
2
0.5/2
0
0
0 28
0.5/2
2
0.5/2
2
0
0
ISA
1.080
1.920)
1.690
1.650
1.430
1.000
1.250
1.720
1.240
1.680
1.620
1.120
1.460
1.770
0.23
1.19
2.37t
2.52t
1.28
1.53
i mmui lyKJAJLju
~ IgM _
0.254
0.227
0.274
0.305
0.165
0.312
0.300
0.233
0.310
0.187
0.237
0.241
0.270
0.300
0.241
0.250
0.252
NO ANIMALS
0.384'
3.379T
0.4971
ISG1
4.60
5.57
6.10
6.40
7.25t
5.80
7.80t
6.70
6.50
3.44
4.44
5.85
7.60
8.70
8.75
7.60
6.80
AVAILABLE
8.70
7.60
4.841
.^7
6.70
8.08
6.15
7.22
5.88
8.22
8.94
6.62
7.10
4.111
6.19
6.62
9.22
9.49t
7.31
5.98
5.56
6.86
7.14
5.69
Arrows indicate either a significant increase or decrease in the
serum immunoglobulin concentration relative to that of filtered-
air controls bled at same time.
IIT RESEARCH INSTITUTE
1-13
-------
Table 1-6
EFFECT OF EXPOSURE TO NITROGEN DIOXIDE ON IMMUNOGLOBULINS
CONCENTRATION IN SERA OF VACCINATED MICE
NO, , ppm
3 Months
Pre-Vacc.
0.5/2
2
0.5/2
0
2
0
0
0
2
0
0
0.5/2
0.5/2
2
0
2
0.5/2
0
2
0.5/2
0
0
2
0.5/2
0
2
0.5/2
0
Post-
Vacc.
.0
0
0.5/2
0
2
2
0.5/2
0
2
0.5/2
2
0.5/2
0
0
0
2
.
0.5/2
0
0.5/2
2
0
2
0
0.5/2
0
0.5/2
2
Number
of
Assays
68
59
54
60
56
61
42
61
56
42
61
53
69
59
60
56
69
42
59
54
61
60
56
68
42
59
54
61
Immunoglobulin, mg/ml
IG Mean
IgA 0.8293
0.8613
0.9183
0.936ab
1.032bc
1.073bc
1.163C
IgM 0.2073
0.217ab
0.217ab
0.237bc
0.244bc
0.246bc
0.263C
IgGi 5.023
1 5.17ab
5.37ab
5.4iab
6.2lbc
6.88C
6.94C
IgG2 5.29a
5.323
5.9lb
5.98b
6.15b
6.26b
6.36b
95% C.L.
0.755-0.906
0.784-0.954
0.829-1.018
0.851-1.032
0.936-1.147
0.973-1.187
1.032-1.315
0.188-0.227
0.196-0.237
0.194-0.244
0.217-0.259
0.219-0.267
0.225-0.267
0.241-0.288
4.37-5.74
4.47-6.02
4.70-6.11
4.57-6.46
5.37-7.23
5.88-8.06
5.97-8.13
4.95-5.69
4.95-5.72
5.56-6.33
5.50-6.51
5.72-6.59
5.85-6.78
5.95-6.86
N02 Exposure groups are listed in the order of increasing
immunoglobulin concentration.
Means with a common superscript, within each immunoglobulin
class, are not significantly different at P < 57..
Exposure to N02 did not influence the concentration of IgA.
The exception was the group of mice maintained in filtered air
before the vaccination and exposed to the 0.5/2 ppm N02 atmos-
phere after the vaccination, which showed a significant increase
in IgA concentration.
The IgM data indicated that concentrations of this immuno-
globulin in all groups of mice exposed to N02 were higher than
that in control mice maintained in filtered air. Statistically
significant increases in IgM levels over the control groups were
observed in groups of mice subjected to: 0-2 ppm N02, 0.5/2-
0.5/2 ppm N02, 0.5/2-0 ppm N02 and 2-0 ppm N02. Moreover, IgM
concentrations in the groups of mice exposed to 2 ppm for 3 months
before vaccination then maintained in filtered air was significantly
higher than in the 2-2 ppm N02 and 0-0.5/2 ppm N02 exposure groups.
The IgGiand IgG2 levels showed a similar pattern in that
the control group had the lowest mean concentration ovei the 28-
week period. The three groups that showed significantly higher
IgG-L concentrations than the controls were 2-0 ppm N02, 0.5-2-
0.5/2 ppm N02, and 0-2 ppm N02. In all but the group of mice
exposed continuously to 2.0 ppm N02, IgG2 concentrations increased
significantly. The 0.5/2-0.5/2 ppm N02, 0-2 ppm N02, and 2-0 ppm
NOo exposure groups showed a significant elevation in this immuno-
globulin subclass as well.
It is interesting that continuous exposure to 2 ppm N02
before and after vaccination did not significantly alter the
levels of any immunoglobulin class. However, exposure of 0-2 ppm
N02, 2-0 ppm N02, and 0.5/2-0.5/2 ppm N02 resulted in significantly
elevated IgM, IgG^ and IgG2 levels. The common factor shared
by these three groups was the change from one environment to
another.
1-14
III RESEARCH INSTITUTE
1-15
-------
Studies of immunoglobulin response Co influenza infection
or vaccination have concentrated on the humoral response of IgM
and IgG, considered to be responsible for, respectively, comple-
ment fixation (CF) and hemagglutination titers (HI) (Daugharty,
et. al, J, Immunol. 109. 849, 1972). Increased levels of serum
IgA have recently been identified in the monomeric form, after
influenza infection or vaccination. Thus, all three immuno-
globulins appear to play a significant role in the host's response
to influenza infection or vaccination.
Our study showed that mice exposed to NO-^ before or after
vaccination produced levels of IgM, IgGi, IgG2 higher than those
in control animals. More specifically, mice consistently showing
elevated levels of these immunoglobulins were those exposed to
air for 3 months before vaccination and then to 2 ppm NO^, those
exposed to. 2 ppm NC>2 for 3 months before vaccination then to
filtered air, and those exposed continuously to 0.5 ppm N0£
with daily peaks of 2 ppm NC>2 before and after vaccination. In
each case the concentrations of NO^ were varied, while exposure
to fixed concentrations of NC>2 before and after vaccination,
even at 2 ppm N02, did not result in elevation of any immuno-
globulin above control levels. Increase in immunoglobulins can
be postulated to affect a protective immune response adversely
since high levels of circulating IgG have been shown to inhibit
the production of specific antibody synthesis (R. Waldman, per-
sonal communication, 1973). Indeed, these three groups of mice,
when challenged with live airborne influenza A2/Taiwan virus,
exhibited the highest mortality rate at 16 and 28 weeks after
vaccination.
4.
Infectious Challenge
Vaccinated mice and nonvaccinated mice injected with saline
were challenged with airborne A2/Taiwan influenza virus at 4,
16, and 28 weeks after vaccination. Comparisons between ob-
served frequencies of mortality were made using the chi-square
III RESEARCH INSTITUTE
1-16
test with a 2 x 2 contingency table, and corrections for
continutiy. The Student t-test was used for statistical eval-
uation of the lung lesion data.
a. Four Weeks after Vaccination. The vaccine appeared
to afford satisfactory protection against the infectious challenge
in mice challenged with airborne influenza virus 4 weeks after
vaccination (Table 1-7). Because of the low challenge dose, only
15% mortality was observed in the nonvaccinated mice. Thus,
the significance of the protection provided by vaccination could
be ascertained only for
• mice continuously exposed to 0.5/2 ppm of NC>2,
• mice held in filtered air before vaccination then
exposed to 2 ppm of NC>2 > and
« mice exposed to 0.5/2 ppm NC>2 before vaccination
then maintained in filtered air.
As Table 1-8 shows, lung lesion scores. confirm the mortality data.
Although the differences in mortality rates within the vaccinated
and nonvaccinated groups were not significant, lung lesions,
which serve as a highly sensitive indicator of influenza infection,
were significantly more severe in several groups of mice. Within
the group of vaccinated mice challenged with the infectious
virus, a significant increase in lung lesions was observed in mice
exposed to 0.5/2 or 2 ppm of NC>2 before vaccination and to fil-
tered air after the vaccination. Nevertheless, in all instances
the vaccine provided sufficient protection so that, irrespective
of the experimental exposure conditions, the extent of lung
lesions was significantly lower in vaccinated than in nonvaccinated
mice.
IIT RESEARCH INSTITUTE
1-17
-------
Table 1-8
LUNG LESIONS IN MICE EXPOSED TO NITROGEN DIOXIDE AND CHALLENGED WITH
INFLUENZA VIRUS 4, 16 AND 28 WEEKS AFTER VACCINATION
N02, ppm
3 Months
Pre-Vacc.
0
0.5/2
2
0
0
0.5/2
2
Post-
Vacc.
0
0.5/2
2
0.5/2
2
0
0
4
Saline
1.95
3.20+
2.37
2.05
3.00+
3.40+
2.90
Lung Lesion Scores
Weeks
Vaccine
0.30*
0.35*
0.35*
0.42
0.44*
1.15+
1.21+
16
Saline
4.80
4.60
4.60
4.27
5.00
4.53
4.13
Weeks
Vaccine
1.27*
2.27*
1.60*
1.92
2.40+
1.40*
2.60+
28
Saline
4.88
4.65
4.83
5.00
4.60
4.30
4.71
Weeks
Vaccine
2.40*
2.92*
3.00*
1.90*
3.47
2.60*
3.80+
a Each score represents the mean lung lesion score of 14 to 20 mice.
Vc
Significant differences between vaccinated and corresponding non-
vaccinated mice.
Significant differences within vaccinated or nonvaccinated groups when
compared with mice held in filtered air.
Table 1-7
MORTALITY RATE IN MICE EXPOSED TO NITROGEN DIOXIDE AND CHALLENGED WITH
INFLUENZA VIRUS 4, 16 AND 28 WEEKS AFTER VACCINATION
NO, , ppm
3 Months
Pre-Vacc .
0
0.5/2
2
0
0
0.5/2
2
Post-
Vacc.
0
0.5/2
2
0.5/2
2
0
0
4 Weeks
Saline
D/T
3/20
9/20
1/19
3/20
7/20
9/20
7/20
7.
15
45
5
15
35
45
35
Vaccine
D/T
0/20
0/20
0/20
0/19
0/18
2/20
3/19
%
0
0*
0
0
0*
10*
16
16 Weeks
Saline
D/T
14/15
13/15
13/15
10/15
15/15
13/15
11/15
7.
93
87
87
67
100
87
73
Vaccine
D/T
1/15
4/15
1/15
3/15
4/15
1/15
6/15
Z
7*
27*
7*
20*
27*
7*
40
28 Weeks
Saline
D/T
25/26
18/20
11/12
5/5
13/15
17/23
12/14
/„
96
90
92
100
87
74
86
Vaccine
D/T
9/25
6/12
7/15
2/10
9/15
8/25
10/15
7.
26*
50*
47*
20*
60
32*
67
Significant differences between vaccinated and corresponding nonvaccinated mice.
-------
b. Sixteen Weeks after Vaccination. At 16 weeks after
vaccination, the vaccine exerted protection in all groups except
mice exposed to 2.0 ppm N02 before vaccination then held in
filtered air (Table 1-7). All vaccinated mice challenged with the
virus showed significantly less severe lung lesions than the
corresponding non-vaccinated control mice (Table 1-8). Within the
vaccinated groups, mice held in filtered air before vaccination
then exposed to 2 ppm NC>2, or those exposed to 2 ppm NC>2 before
vaccination then maintained in filtered air for 16 weeks had more
severe influenza lung lesions than the vaccinated mice held con-
tinuously in filtered air.
c. Twenty-eight Weeks after Vaccination. The vaccine
was protective in all but two groups of mice challenged with
virus 28 weeks after vaccination (Table 1-7). The two groups were
mice held in filtered air prior to vaccination then exposed to
2 ppm NC>2 and mice exposed to 2 ppm NC>2 before vaccination then
maintained in filtered air for 28 weeks before the infectious
challenge.
Vaccinated mice held in these two environmental conditions
had lung lesion scores that were similar to the corresponding
non-vaccinated mice (Table 1-8), an indication of increased sus-
ceptibility to infection. Within the vaccinated groups, only
mice held in 2 ppm NC>2 for 3 months before vaccination then main-
tained in filtered air for 28 weeks and challenged with the in-
fectious virus had more severe lung lesions than vaccinated mice
held continuously in filtered air. Thus, this group of mice,
when challenged at 4, 16, or 28 weeks after vaccination, appeared
to be more susceptible to infection as measured by mortality rates
and lung, lesion scores.
Mice surviving the virus challenge at 16 and 28 weeks after
vaccination were exsanguinated and the sera were assayed for
HI antibody to determine the anamnestic response. Vaccinated
mice in both the control and experimental groups showed a
IIT RESEARCH INSTITUTE
1-20
consistent and excellent anamnestic response to the influenza
virus infection (Table 1-9). The primary immune response is that
shown by nonvaccinated mice injected saline then challenged with
influenza virus.
5.
Lung Edema
The extent of lung edema as measured by the wet-to-dry lung-
weight ratios was examined at the various period. In general,
the differences in mean lung edema ratios between mice exposed to
filtered air and those exposed to N02 were not significant (Table
1-10), There were some marked differences in edema ratios among
the various groups of mice exposed to N02 but no pattern was
evident. Thus, after approximately 10 months exposure to NC>2,
no significant differences in the lung edema ratios were found
between mice exposed to NC>2 and filtered air.
6. Histopathology
Histopathological examination of lungs from mice vaccinated
against influenza two weeks after vaccination revealed a mild
subacute. to acute pneumonitis with slight to mild bronchiolar
epithelial hyperplasia. Many had mild peribronchiolar lymphocytic
accumulations reflecting a baseline chronic murine pneumonia
(Figure 1). Mild peripheral emphysema was also present in some
of the mice. The various N(>2 exposure conditions did not appear
to influence the type or the extent of pathological changes.
The lungs of mice vaccinated against influenza examined four
weeks after the vaccination showed similar pathology. A slight
subacute interstial pneumonitis was present with baseline chronic
murine pneumonia. These lungs were in a marked contrast to those
of mice challenged with A2/Taiwan influenza virus 2 weeks after
the vaccination and killed 2 weeks later. A mild to moderate
amount of focal parabronchial and peribronchiolar bronchoalveolar
proliferation (pulmonary adenomatosis, alveolar fetalization,
bronchiolar adenomatoid lesion) was present (Figure 2). The
alveolar lining cells were hyperplastic and gave the appearance
IIT RESEARCH INSTITUTE
1-21
-------
o
I
Table I-10
WET:DRY LUNG RATIO IN MICE EXPOSED TO NITROGEN DIOXIDE
wu_ , ppm
J Months
Pre-Vacc,
0
0.5/2
2
0
0
0.5/2
2
Post-
Vacc.
0
0.5/2
2
0.5/2
2
0
0
~2
4.53
4.54
4.70
4.78
4.66
4.58
4.46
4
4.68
4.50
4.54
4.52
4.39
4.58
4.87
Ratio,
Weeks
8
4.64
4.55
4.65
4.48
4.64
4.5.0
4.54
wet-to-dry lung weight
after Vaccination:
12
4.11
4.79*
3.88
4.72
4.08
4.44
4.60
16
4.70
4.40
5.06
4.43
4.55
4.72
4.64
20
4.53
4.45
4.85
4.54
4.60
4.38
4.48
24^
4.52
4.21
4.25
4.35
4.39
4.26
4.40
28
4.07
4.26
4.18
(a)
4.10
3.96
3.97
*
Significant differences when compared with mice exposed to filtered air.
'Animals not available.
Table 1-9
MEAN HI TITERS IN SURVIVING MICE EXPOSED TO NITROGEN DIOXIDE
AND INFLUENZA VIRUS AT 16 AND 28 WEEKS AFTER VACCINATION
N02' ppm Reciprocal HI Titer
3 Months
Pre-Vacc
0
0.5/2
2
0
0
0.5/2
2
Post-
Vacc .
0
0.5/2
2
0.5/2
2
0
0
16 Weeks
Saline
8
288
28
28
N
36
(1)
(2)
(2)
(5)
(0)
.D.*
(4)
Vaccine
243
277
294
309
256
300
272
(14)
(11)
(14)
(12)
(11)
(11)
(9)
28 Weeks
Saline
24
24
24
<16
136
24
(1)
(2)
(D
(0)
(2)
(6)
(2)
Vaccine
151
99
155
101
99
267
128
(16)
(6)
(8)
(8)
(6)
(17)
(5)
Number of surviving mice shown in parentheses.
Not determined.
-------
FIGURE 1: Mild parabronchial lymphocytic accumulations indicative
of chronic murine pneumonia in lungs of vaccinated
mouse exposed for 3-1/2 months to 0.5 ppm NC>2 with
daily 1 hr peaks of 2 ppm N02- 64x
FIGURE 2:
Moderate local peribronchial and parabronchial broncho-
alveolar proliferation in lungs of vaccinated mouse
challenged with live influenza virus. The mouse was
exposed for 3 months to 0.5 ppm N02 with daily 1 hr
peaks of 2 ppm N02 before vaccination and held for
1 month in filtered air after vaccination. 48x
of a cuboidal to columnar epithelium. Some of the cells possessed
well-developed cilia. Cellular atypia was present in some of these
proliferative lesions, but the extent was not sufficient for them
to b£ considered anaplastic (Figure 3). Subacute interstitial
pneumonitis, slight marginal emphysema, and slight chronic murine
pneumonic changes were also present. The lungs of nonvaccinated
mice challenged with virus and examined at 4 weeks, had the same
type and extent of pathological changes. Differences in the extent
or type of pathological changes observed in the lung tissues did
not appear to be related to the NC>2 exposure conditions.
Lungs of vaccinated mice examined 16 and 28 weeks after
vaccination had minimal changes, which were comparable to those
observed in the lungs of vaccinated mice examined at 2 and 4 weeks.
Alveologenic adenomas which are frequently found in older mice and
are considered spontaneous tumors were present in two mice examined
at 28 weeks.
Mild to severe focal peribronchial bronchoalveolar proliferative
lesions were present in both vaccinated and nonvaccinated mice
surviving the infectious virus challenge at 16 and 28 weeks after
vaccination and killed two weeks later. Some areas of the pro-
liferative reaction had moderate cellular atypia and were suggestive
of carcinoma. A mild to marked peribronchial subacute inflammatory
infiltrate, and moderate bronchial and bronchiolar epithelial hyper-
plasia (Figure 4) were present. No differences could be seen between
nonvaccinated and vaccinated mice or the various N02 exposure groups.
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FIGURE 3:
Proliferative lesions and cellular atypia suggestive
of carcinoma in lung of vaccinated mouse exposed to
2 ppm N(>2 for 4 months and challenged with live
influenza virus. 95x ' x
FIGURE 4: Mild to marked peribronchial subacute inflammatory
infiltrate and moderate bronchial and bronchiolar
epithelial hyperplasia in lung of vaccinated mouse
challenged with live influenza virus, exposed to
filtered air. 122x
D. Summary
Swiss albino mice were continuously exposed to either 2 ppm
N02, 0.5 ppm NO, with daily 1-hr peaks of 2 ppm N02 5 days a week,
or filtered air. After a 3 month exposure the mice were vaccinated
with A2/Taiwan influenza vaccine and the exposures to the various
environmental conditions continued for an additional 28 week period.
At various time intervals sera were obtained for HI and SN antibody
and immunoglobulin assays. In addition, at 4, 16 and 28 weeks
after vaccination groups of mice were challenged by the respiratory
route with airborne live A2 /Taiwan influenza virus. The mortality
rates, lung lesion scores, and histopathological changes in lung
tissue were observed.
Throughout the experiment all groups of mice gained weight
consistently irrespective whether or not exposed to N02- Similarily,
the formation and decline of HI antibody titers;did not appear to
be influenced by the environmental exposures. However, within 2
weeks after vaccination a significant depression of SN antibody
titers was observed in mice continuously exposed to 0.5 ppm N02
with 2 ppm N02 peaks and those held in filtered air before vaccin-
ation and 2 ppm NC>2 or 0.5 ppm NC>2 with 2 ppm NC>2 peaks after the
vaccination. Furthermore, at the same time period the SN isero-
conversion rate among mice continuously exposed to 0.5 ppm N09
with 2 ppm N02 peaks was 07, while in those exposed to filtered air
it was 100%. After the initial 2 week period the SN titers did
not differ significantly between the various experimental groups.
Immunoglobulin levels of mice exposed to N02 for 3 months
prior to vaccination differed significantly from those held in
filtered air. There was a significant decrease in serum IgA and
a significant increase in IgG^, IgG2, and IgM levels. During the
28 week period following vaccination, exposure to N02 did not in-
fluence the concentration of IgA. However, IgM, IgG]_ and IgG2
levels in all mice exposed to N02 were higher than in mice main-
tained in filtered air. More specifically, mice consistently
showing statistically higher immunoglobulin levels were those
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exposed to filtered air for 3 months then to 2 ppm N02,
to 2 ppm N02 for 3 months then to filtered air,
Peaks of
These three groups of mice also exhibited the highest mortality
rates and severest pulmonary lesions when challenged with live
airborne A2/Taiwan influenza virus at 16 and 28 weeks after
vaccination.
Lung tissues of mice challenged with the live influenza virus
and sacrificed 2 weeks later showed moderate to severe broncho-
alveolar proliferative reaction. Small to large focal areas of
parabronchial or peribronchiolar bronchoalveolar proliferation
were present with ciliation and slight cellular atypia in some
portions. Subacute interstitial pneumonitis, slight marginal
emphysema, and chronic murine pneumonia were also present. The
bronchoalveolar proliferation was absent from the lungs of vacc-
inated animals not subsequently challenged with virus. Variation
of N02 treatment did not appear to influence the extent of the
proliferative inflammatory reaction, nor lung edema.
Results of these studies suggest that fluctuations in
environmental conditions represent a more significant factor in
immune response than exposure to a single stressful atmosphere.
Continuous exposure of mice for approximately 10 months to 2 ppm
N02 did not appear to influence formation of antibodies or levels
of immunoglobulins, nor their resistance to subsequent respiratory
challenge with live influenza virus. Conversely, continuous ex-
posure to 0,5 ppm N02 with daily 1-hr peaks of 2 ppm NO2 appeared
to depress the ability to form SN antibodies and significantly
altered the levels of IgM, lgG1 and IgG2 immunoglobulins. Further-
more, these mice developed a more severe infection as reflected
by increased mortality rates upon challenge with live influenza
virus.
II. IMMUNE RESPONSE IN MICE DURING A 4-WEEK EXPOSURE TO N02
A. Introduction
This study was conducted to examine the immune response of
vaccinated mice during a one month exposure to low levels of
nitrogen dioxide (N02). Mice were held in ambient air for 6 weeks,
vaccinated with purified influenza vaccine and then exposed to
N02. Parameters measured were hemagglutination-inhibition (HI)
and serum neutralizing (SN) antibody formation, serum immuno-
globulin levels, and mortality rates in mice challenged with live
influenza virus,
B.
Materials and Methods
Animals. Six-week-old specific-pathogen-free male Swiss
albino mice, CD-I strain, were obtained from Charles River Lab-
oratories. After a six-week quarantine, the 3 month old mice
were placed in the environmental chambers. During the exposures,
the mice were removed from the chamber for one hour three times
a week for maintenance. Clean cages were provided once a. week
and food and water were provided ad libitum.
Influenza Virus. Mouse-adapted influenza A2/Taiwan/l/64
virus awas passaged several times in mice and 207= lung suspension
of the virus was used for all infectious challenges. Prior to
use, the virus was identified by use of specific antiserum ob-
tained from the National Institutes of Health.
Vaccine. Chick embryo A2/Taiwan/l/64 influenza vaccine
(Zonomune), Lot No. BP0549, was supplied by Eli Lilly and Company,
Indianapolis. Mice were vaccinated by a single subcutaneous
injection of approximately 279 CAA units in 0.1 ml vaccine.
IIT RESEARCH INSTITUTE
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HI and SK Titrations. Tests were performed in duplicate,
by the microtiter method in disposable V-plates (Cooke Engineering
Co., Alexandria, Va.) described by Davenport and Minuse (Diagnostic
Procedures for Viral and Rickettsial Diseases, 3rd ed. Am. Public
Health Assoc., New York, 1964). In all tests, 17= chicken red blood
cells were used.
Four hemagglutinating units of antigen were used for the
HI test. All antisera were heat-inactivated at 56°C for 30 min
and treated with trypsin-periodate to remove nonspecific inhibitors
of hemagglutination.
The protocol used for the SN test was similar to that described
in the USPHS Requirements (Division of Biologies Standards, 6th
rev., 1947). Sera were heat-inactivated at 56°C for 30 min,
serially diluted, and incubated with an equal volume of influenza
virus for 1 hr at 4°C. The serum-virus mixture was then tested
in 10-day-old embryonated chicken eggs. The eggs were inoculated
by the allantoic route with 0.1 ml of the virus-serum mixture,
incubated at 37°C and harvested when an EIDjg (507= egg infectious
dose) dose of 32 to 320 was attained as indicated by hemagglutination
of the virus control. The EID5Q dose was determined by parallel
infectivity tests in eggs by using a 0.1-ml virus-saline mixture.
Phosphate buffered saline (PBS), PBS plus normal mouse serum, and
normal mouse serum plus virus were inoculated into eggs as
controls.
Immunoglobulin Concentration. Quantitative radial immuno-
diffusion plates for mouse immunoglobulins IgA, IgG^, IgG2, and
IgM were procured from Meloy Laboratories, Inc., Springfield,
Virginia. Reference standards obtained from pooled sera of normal
mice were assayed daily, in duplicate, to provide quality control.
Duplicate serum samples were placed in preformed wells, the plates
incubated at 22°C for 18 hr and the radial diffusion diameters
measured using a Bausch & Lomb 7x lens. Duplicate radial diffusion
diameters were recorded for each of the pooled serum sample;
consisting of seven to 10 pooled samples at each experimental point.
Ill RESEARCH INSTITUTE
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Nitrogen Dioxide Exposure. To maintain the control and
experimental mice under similar conditions, three identical
aluminum-lined chambers (4 x 6 x 6.5 ft) were used for the ex-
posures. Randomly selected mice were housed in suspended wire
cages, which were periodically rotated to various position on
the cage racks. This assured a thorough and unbiased exposure
to the experimental environment. A minute amount of N(>2 was
continuously passed from a cylinder through a stainless steel
tube into a glass mixing vessel where it was diluted and mixed
with charcoal-filtered ambient air. The mixture was then passed
into the N02 exposure chambers at a rate of 20 changes per hour.
The same air flow pattern was maintained in the control chamber
where the charcoal-filtered ambient air was used. To verify the
homogeneity of NC>2, air samples were taken from different sections
of the chambers and the NC>2 concentration determined and calculated
by the Saltzman method. A Mast gas analyzer was used for continuous
NC>2 monitoring. The mean temperature in the chambers was ?4° ~'+ 1°C.
Scoring Pulmonary Lesions. The extent of pulmonary lesions
was expressed as the percentage of the total lung consolidated.
A score of 1 represented 257, lung consolidation, 2 = 507=, 3 = 757.,
4 = 1007o, and a score of 5 represented animals that died during
the experiment.
Aerosol Challenge. Infectious respiratory challenge was
conducted in a 350-liter plastic aerosol chamber (60 x 60 x 95 cm)
installed within a microbiological safety cabinet. A University
of Chicago Toxicity Laboratory type atomizer was used to produce
a viral aerosol with a majority of <5|i-mass-median-diameter par-
ticles. The microbial suspensions were fed to the atomizer by a
50-ml syringe activated by a motor-driven piston delivering the
suspension at a rate of 0.4 ml/min. Filtered air was supplied
to primary and secondary inlets of the atomizer at a flow rate of
33 liters/min. The chamber was maintained at 78 + 67= RH and
24 + 1°C.
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The aerosol was sampled with an all-glass impinger (AGI-30)
containing PBS with 0.27. bovine serum albumin as a collecting
fluid. The inhaled dose was estimated on the basis of the con-
centration of the virus per liter of air, respiratory minute
volume of the mice, and the duration of exposure to the aerosol.
For the infectious challenge, groups of mice were placed in
the chamber and exposed to the aerosol for 5 to 6 min. After the
challenge, the mice were air-washed for 10 min, removed from the
aerosol chamber and held for 14 days in filter-capped cages in a
clean-air, isolated animal room.
Experimental Protocol. After vaccination the mice were
exposed continuously to one of the following three environmental
conditions :
• 2.0 ppm of N02 (2 ppm N02>
• 0.5 ppm of NC>2 with daily 1-hr peaks of 2.0 ppm of
N02 for 5 days /week (0.5/2 ppm N02)
• filtered air (0 ppm
Groups of 14 to 20 mice were killed at 1, 2, and 4 weeks after
vaccination. Sera from two mice were pooled and assayed for HI
and SN antibodies and for immunoglobulin levels. To determine
the protective effect of the vaccine, as measured by mortality
rates and lung lesion scores, groups of 20 mice were challenged
with A2 /Taiwan influenza virus at 1, 2, and 4 weeks after the
vaccination.
Statistical Analysis. As appropriate, the experimental
results were subjected to statistical analysis and the significance
of the observed differences was reported at the <57<, probability
level. The immunoglobulin level data were analyzed by a two-way
analysis of variance and a multiple range test was used to
determine the ranking and significance of the observed differences.
The significance of the differences in mortality rates was determined
by the normal approximation with correction for continuity (K. S.
Brownlee, Statistical Theory and Methodology in Science and
Engineering, p. 121, 1960, John Wiley and Sons, New York). This
method is equivalent to the chi-square test applied with a 2 x 2
contingency table, except for the use of corrections for continuity.
The Student t-test was used for statistical analysis of the lung
lesion scores.
C.
Results
Antibody Formation. The HI and SN antibody responses were
noted in all mice within one week after vaccination, but no sig-
nificant, 4-fold, differences in the titers were observed through-
out the 4 week exposure period (Table II-l). Similarly there were
no significant differences in seroconversion rates, although some
depression of seroconversion was observed in mice held in N02
atmospheres for 1 week. With some discrepancy, the overall pattern
of antibody response was similar to that observed during the
first month of the previously reported long-term chronic exposure
to N02. The inconsistency of the observations could be in part
ascribed to the vaccine per se. The vaccine used in this experi-
ment was obtained at a different time. Although the label in-
dicated that the vaccine was from the same lot as the one used
during the long-term chronic exposure studies, nevertheless it
appeared to be markedly more immunogenic. An additional factor
contributing to the discrepancy was the pre-vaccination exposure.
In the long-term chronic studies the mice were exposed to 2 ppm
N02, 0.5/2 ppm NC>2 or filtered air for 3 months before vaccination.
In this study all mice were exposed to filtered air for 1 1/2 months
before the vaccination.
NT RESEARCH INSTITUTE
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IIT RESEARCH INSTITUTE
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Table II-l
MEAN HI AND SN RECIPROCAL TITERS AND SEROCONVERSION RATES
IN VACCINATED MICE EXPOSED TO N02
M a
rH O
I I
Antibody Tlter
N02,
PPm
0
0.5/2
2
lc
37
27
26
Hia
2
30
46
40
4
54
56
105
r
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23
19
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71
33
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1
90
70
60
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2
100
100
100
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100
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100
86
80
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78
100
100
4
100
100
100
Four antigen units of egg grown A2/Taiwan influenza virus used in
the HI teat.
Tested against 23-100 £1050 units.
c Weeks of exposure to N02 and after vaccination.
-------
Table II-2
SERUM IMMUNOGLOBULIN CONCENTRATIONS IN
VACCINATED MICE EXPOSED TO N02
N02,
0
0
0.5/2
2
0
0.5/2
2
0
0.5/2
2
Weeks
Post-Vacc.
0
1
2
4
Immunoglobulin, rag/ml
0.413
0.368
0.470T
0.366
0.416
0.515*
0.455
0.400
0.540*
0.8801*
0.094
0.338*
0.2741
0.286
0.255
0.195
0.248
0.255
0.195
0.252
1.18
3.12*
1.99
1.251
1.11
0.92
1.02
1.27
1.58
1.55
3.17
5.26*
3.92
2.44j*
2.77
2.51*
2.45
3.41
3.25
3.41
Star indicates significant change from pre-vaccination
level.
Arrow indicates significant change from control mice
held in filtered air.
over the 28 week period. The 4 week exposure to N02 resulted
in an elevation of IgA but an initial depression of IgM, IgG-^, and
IgG2- These differences are most likely due to the varying time
of sampling whereby this experiment reflects the immunoglobulin
concentration changes during the initial 4 weeks after vaccination.
In addition, the animals used in the acute study were 2 to 4 weeks
younger than at the initiation of the exposure than used in the
chronic NOo study.
Infectious Challenge. Vaccinated mice and non-vaccinated
mice injected with saline were challenged with airborne A2/Taiwan
influenza virus at 1, 2, and 4 weeks of exposure to N02- The
mortality rates and lung lesion scores are summarized in Tables
II-3 and II-4, respectively.
The vaccine afforded excellent protection against the
infectious challenge under all exposure conditions and no significant
differences were noted in mortality rates when mice held in N02
were compared to the control mice exposed to filtered air through-
out the 4 week period. Similarily, the lung lesions scores were
significantly lower in all vaccinated mice when compared to non-
vaccinated mice. At 4 weeks after vaccination, the lung lesion
scores were significantly less severe in mice exposed to N0£ than
those exposed to filtered air. These results are probably due to
the very high efficacy of the vaccine or to an experimental variable.
This was not observed in non-vaccinated mice nor in any other group
of mice included in this experiment.
Mice surviving the virus challenge at 1, 2, .and 4 weeks after
vaccination were exsanguinated and the sera were assayed for HI
antibody to determine the anamnestic response. The various groups
of vaccinated mice showed a consistent ananmestic response to A2/
Taiwan influenza virus infection, with no effect of the NOg ex-
posure being apparent.
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Table II-4
LUNG LESIONS IN MICE CHALLENGED WITH A2/TAIWAN
INFLUENZA VIRUS
7 S
N02)
ppm
0
0.5/2
2
Lung Lesion Scores *• '
1 Week 2 Weeks
Saline Vaccine Saline Vaccine
4.40 0.85* 3.70 0.87*
4.85 0.70* 4.25 0.80*
4.20 1.40* 3.80 0.25*
4 Weeks
Saline Vaccine
3.80 1.60*
3.83 0.15*+
4.15 0.26*+
(a)
Each score represents the mean lung lesion score of 14
to 20 mice.
Significant difference between vaccinated and corresponding
non-vaccinated mice.
Significant difference within vaccinated group when
compared to mice held in filtered air.
Table II-3
MORTALITY RATE OF MICE CHALLENGED WITH
A2/TAIWAN INFLUENZA VIRUS
Exposure to NO?. Weeks
N02,
ppm
0
0.5/2
2
1
Saline
D/T
15/19
18/20
13/20
79
90
65
Vaccine
D/T
0/20
0/20
1/20
7.
0*
0*
5*
2
Saline
D/T
9/20
13/20
12/20
la
45
65
60
Vaccine
D/T
1/16
1/20
0/20
7>
6*
5*
0*
4
Saline
D/T
12/20
12/18
14/20
7«
60
67
70
Vaccine
D/T
2/12
0/20
0/20
%
17*
0*
0*
Significant differences between vaccinated and corresponding non-vaccinated
mice.
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Summary. Mice were vaccinated with A-j/Taiwan influenza
vaccine and exposed for 4 weeks to either 2 ppm of NC^s 0,5 ppm
N02 with 1-hr peaks of 2 ppm N07 for 5 days per week, or to filtered
air. The differences among the HI and SN antibody titers between
control and experimental mice were not significant. There was an
elevation in the levels of IgA, and an initial depression of IgM,
IgGl and IgG2 concentration. No statistical differences were
noted in mortality rates or lung lesion scores when infected mice
exposed to NC>2 were compared to infected mice held in filtered air.
HI antibody titers in mice surviving virus challenge indicated that
NC>2 exposure had no effect on the anamnestic response of vaccinated
mice.
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III.
EFFECT OF CHRONIC EXPOSURE TO N09 ON RESISTANCE TO
K. PNEUMONIAS
A.
Introduction
Limited studies were conducted to determine changes in
resistance to bacterial pneumonia associated in the fluctuation
in concentrations of nitrogen dioxide (N02). Mice were held in
a selected environment for 3 months and then re-exposed to the
same environment or transferred to another environmental condition.
At various time intervals the mice were challenged with Klebsiella
pneumoniae, returned to the appropriate environmental condition
and mortality rates were determined over a 14 day period.
B. Materials and Methods
Animals. Four-week-old specific-pathogen-free male Swiss
albino mice, CD-I strain, were obtained from Charles River Lab-
oratories. After a two-week quarantine, the mice were placed in
the environmental chambers and held for two days before initiation
of the exposures. During the exposures, the mice were removed
from the chamber for one hour three times a week for maintenance.
Clean cages were provided once a week and food and water were
provided ad libitum.
Nitrogen Dioxide Exposure. To maintain the control and
experimental mice under similar conditions, three identical
aluminum-lined chambers (4 x 6 x 6.5 ft) were used for the ex-
posures. Randomly selected mice were housed in suspended wire
cages, which were periodically rotated to various positions on
the cage racks. This assured a thorough and unbiased exposure
to the experimental environment. A minute amount of N02 was con-
tinuously passed from a cylinder through a stainless steel tube
into a glass mixing vessel where it was diluted and mixed with
charcoal-filtered ambient air. The mixture was then passed into
the N(>2 exposure chambers at a rate of 20 changes per hour.
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The same air flow pattern was maintained in the control chamber
where the char coal-filtered ambient air was used. To verify
the homogeneity of N02, air samples were taken from different
sections of the chambers and the NC>2 concentration determined and
calculated by the Saltzman method. A Mast NC>2 gas analyzer was
used for continuous monitoring. The mean temperature in the
chambers was 24° + 1°C.
Klebsiella pneumoniae. Bacterial challenge was made with
mouse-adapted K. pneumoniae type A, strain A-D. To prepare the
stock culture K. pneumoniae was passaged in mice, isolated from
the heart, and grown in trypticase soy broth for 18 hr at 37°C
in static culture. The bacteria were then streaked onto blood
agar slants, incubated for 18 hr at 37°C, and held at 4°C until
used. For dissemination, bacteria from the slants were grown
in trypticase soy broth at 37°C for 18 hr in static culture.
The concentration of the culture was adjusted by measurement of
transmission on a Spectronic 20 densitometer at 440 pm. Appropriate
dilutions of the culture were used for dissemination to obtain
approximately 207= mortality in control group of mice.
Aerosol Challenge. Infectious respiratory challenge was
conducted in a 350-liter plastic aerosol chamber (60 x 60 x 95 cm)
installed within a microbiological safety cabinet. A University
of Chicago Toxicity Laboratory type atomizer was used to produce
the bacterial aerosol with s majority of <5ta.-mass-median-diameter
particles. The microbial suspensions were fed to the atomizer
by a 50-ml syringe activated by a motor-driven piston delivering
the suspension at a rate of 0.4 ml/min. Filtered air was supplied
to primary and secondary inlets of the atomizer at a flow rate
of 33 liters/min. The chamber was maintained at 78 + 67, RH and
24° + 1°C.
The aerosol was sampled with an all-glass impinger (AGI-30)
containing PBS with 0.2% bovine serum albumin as a collecting
fluid. The inhaled dose was estimated on the basis of the con-
centration of K. pneumoniae per liter of air, respiratory
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minute volume of the mice, and the duration of exposure to the
aerosol.
For the infectious challenge, groups of mice were placed
in the chamber and exposed to the aerosol for 5 to 6 min. After
the challenge, the mice were air-washed for 10 min, removed from
the aerosol chamber and held for 14 days in filter-capped cages
in a clean-air, isolated animal room.
Statistical Analysis. As appropriate, the experimental
results were subjected to statistical analysis and the significance
of the observed differences was reported at the <57o probability
level. The significance of the differences in mortality rates
was determined by the normal approximation with correction for
cintinuity (K. S. Brownlee, Statistical Theory and Methodology
in Science and Engineering, p. 121, 1960, John Wiley and Sons,
New York). This method is equivalent to the chi-squart test
applied with a 2 x 2 contingency table, except for the use of
corrections for continuity.
C.
Results
Mortality rates were determined in mice continuously exposed
for up to 9 months to either 2 ppm N02, 0.5 ppm NC>2 with daily
1-hr peaks of 2 ppm N02, or filtered air. At various time inter-
vals during the exposure groups of 20 mice were challenged with
airborne K. pneumoniae and mortality rates were observed over a
14-day holding period. The results of the experiment summarized
in Table III-l, indicate that there was an increase in mortality
rates among mice exposed to 2 ppm N02 and to a lesser degree
among those exposed to 0.5 ppm N02 with daily 1-hr peaks of 2 ppm
N02- Because of the very limited number of mice available in
this study, replicate experiments were not performed. Thus the
statistical significance of the differences could be ascertained
only for groups exposed to N02 for eight months. However, as
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Table III-l
PERCENT MORTALITY IN MICE EXPOSED TO NOo
AND CHALLENGE WITH K. PNEUMONIAS
N02,
ppm
0
0.5/2
2
N02 Exposure ,
Months
6
5
23
26
7__
65
50
70
B
25
60*
75*
9^
15
15
25
Significant change when
compared to mice exposed
to filtered air (0 ppm N02).
Table III-Z
PERCENT MORTALITY IN MICE CHALLENGED WITH
K. PNEUMONIAE DURING RECOVERY
FROM EXPOSURE TO N(>2
3-Months
Exposure
N02. Ppm
0
0.5/2
2
Filtered Air
Exposure. Months
3 _ 4 5~ F
5 65
15 40
5 50
25
50
55
15
25
15
previously reported from our laboratories chronic exposure to
0.5 ppm NOj reduced the resistance of mice to subsequent challenge
with airborne K. pneumoniae. The limited experiments summarized
in this report confirmed these observations.
Mice exposed for 3 months to 0.5 ppm N02 with 1-hr peaks of
2 ppm N02 and to 2 ppm N02 were held in filtered air for an add-
itional 6 months. During this recovery period groups of 20 mice
were challenged with airborne K. pneumoniae and the mortalities
were observed during a 14-day holding period. The results summ-
arized in Table III-2 indicate no marked differences in mortality
rates, especially when compared to mice exposed to filtered air only.
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IV. EFFECTS OF MANGANESE OB RESISTANCE TO RESPIRATORY INFECTION
A.
Introduct ion
Studies were conducted to determine the effect of acute
exposures of airborne manganese oxide (MnO) or manganese dioxide
(Mn02) on resistance of mice to bacterial or viral respiratory
infections. Initially, exploratory experiments were conducted
to develop aerosolization techniques by means of the Wright Dust
Feeder. Mice were exposed to airborne manganese oxide (MnO) or
manganese dioxide (Mn02) for various time periods, killed, and
the 3ungs were examined microscopically to observe the deposition
and retention of particles.
Thereafter groups of mice were exposed daily for 3 hrs up
to 4 days, to the manganese compounds. The mice were challenged
with influenza virus before exposure and with K. pneumoniae following
the exposure to manganese.
B.
Materials and Methods
Animals. Four week-old specific-pathogen-free male Swiss
albino mice, CD-I strain, were obtained from Charles River Lab-
oratories. After a two-week quarantine period, the mice were
used for the exposures. Throughout the experiments clean cages
were provided once a week and food and water were provided ad
libitum.
Influenza Virus. Mouse-adapted influenza A/PR/8 virus was
passaged several times in mice and 20% lung suspension of the virus
was used for all infectious challenges. Prior to use, the virus
was identified by use of specific antiserum obtained from the
National Institutes of Health.
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Klebsiella pneumoniae. To prepare the stock culture, K.
pneumoniae type A, strain A-D were passaged in mice, isolated from
the heart, and grown in trypticase soy broth for 18 hr at 37°C
in static culture. The bacteria were then streaked onto blood agar
slants, incubated for 18 hr at 37°C, and held at 4°C until used.
For dissemination, bacteria from the slants were grown in trypti-
case soy broth at 37°C for 18 hr in static culture. The concen-
tration of the culture was adjusted by measurement of transmission
on a Spectronic 20 densitometer at 440 um. Appropriate dilutions
of the culture were used for dissemination to obtain approximately
207= mortality in control group of mice.
Aerosol Challenge. Infectious respiratory challenge was
conducted in a 350-liter plastic aerosol chamber (60 x 60 x 95 cm)
installed within a microbiological safety cabinet. A University
of Chicago Toxicity Laboratory type atomizer was used to produce
the microbial aerosol with a majority of <5n.-mass-median-diameter
particles. The microbial suspensions were fed to the atomizer by
a 50-ml syringe activated by a motor-driven piston delivering the
suspension at a rate of 0.4 ml/min. Filtered air was supplied
to primary and secondary inlets of the atomizer at a flow rate of
33 liters/min. The chamber was maintained at 78 + 6% RH and 24 +
1°C.
The aerosol was sampled with an all-glass impinger (AGI-30)
containing PBS with 0.2% bovine serum albumin as a collecting fluid.
The inhaled dose was estimated on the basis of the concentration
of the microorganisms per liter of air, respiratory minute volume
of the mice, and the duration of exposure to the aerosol.
For the infectious challenge, groups of mice were placed in
the chamber and exposed to the aerosol for 5 to 6 min. After the
challenge, the mice were air-washed for 10 min, removed from the
aerosol chamber and held for 14 days in filter-capped cages in
a clean-air, isolated animal room.
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Scoring Pulmonary Lesions. The extent of pulmonary lesions
after challenge with influenza virus was expressed as the percentage
of the total lung consolidated. A score of 1 represented 25% lung
consolidation, 2 = 50%, 3 = 757., 4 = 100%, and a score of 5 repre-
sented animals that died during the experiment.
Exposure to Manganese. Mice were placed in a plexiglass
chamber (120 x 60 x 60 cm; 430 liter capacity) and exposed for
3 hr to MnO or MnC>2 aerosols produced by the Wright Dust Feed
Mechanism (L. Adams Ltd., London, England). The amounts of MnO
or MnC>2 disseminated in the chamber over the 3 hr period were 50
and 76 g, resulting in an aerosol concentration of approximately
110 jig MnO/ml and 168 ng Mn02/ml of air. To ensure uniform
exposure to aerosol, randomly selected mice were housed in wire
cages, which were rotated at least once during the 3-hr exposure
to vary the position within the chamber.
During the aerosol exposure, microscopic slides were placed
in horizontal positions in the chamber for time periods ranging
from 15 to 60 sec. The slides were viewed at lOOOx magnification
and 100 particles were counted to determine the percentage of
particles equal to or less then 5u in diameter. Approximately 5%
of the MnO particles were £5|x diameter, whereas 757., of the Mn02
particles were in this size range.
Statistical Analysis. As appropriate, the experimental
results were subjected to statistical analysis and the significance
of the observed differences was reported at the <5% probability
level. The significance of the differences in mortality rates
was determined by the normal approximation with correction for
continuity (K. S. Brownlee, Statistical Theory and Methodology
in Science and Engineering, p. 121, 1960, John Wiley and Sons,
New York). This method is equivalent to the chi-square test
applied with a 2 x 2 contingency table, except for the use of
corrections for continuity. The Student t-test was used for
statistical analysis of the lung lesion scores.
The relative mean survival rate (RMSR) was calculated
according to the following equation
RMSRd
x B) + (d x L)
Where A is the last day on which any individual mouse was alive;
B is the number of mice surviving A days; d is the last day of
the experiment (14 or 15); L is the number of mice which were
alive on day d; and n is the initial number of mice in the
experimental group. The significance of the differences in the
RMSR values between the experimental and control groups was
assessed bv the Student t-test (2-tailed).
C.
Results
MnO Exposure and Challenge with Influenza Virus. Mice
were exposed to MnO for 3 hr daily, with the first exposure
occurring 24 hr after the challenge with influenza virus. Since
approximately 957.. of the MnO particles were larger than 5a in
diameter, groups of mice were sacrificed to determine whether MnO
particles were present in the lungs. Following three 3-hr exposures,
MnO particles were not seen in the lung upon examination in
transmitted or polarized light microscope. After four 3-hr ex-
posures, a small number of MnO particles was scattered throughout
the lung tissue. Nevertheless, as shown in Table IV-1 increased
mortality rates were observed among mice challenged with the virus
and exposed to MnO.
Mn02 Exposures and Challenged with K. pneumoniae. During
the initial experiments, lungs were excised from the mice immediately
after each exposure to Mn02- Microscopic observation of the lungs
revealed that Mn02 particles were present after a single 3 hr
exposure and the number of particles increased with the number of
the 3-hr exposures to MnC>2. At all time periods, an acute to
subacute septal infiltrate was present, with slight septal edema
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Table IV-1
MORTALITY RATE AMONG MICE CHALLENGED WITH
INFLUENZA VIRUS AND EXPOSED TO MnO AEROSOL
Experimental
Condition
MnO (3)a
MnO (4)
Virus
Virus -* MnO (3)b
Virus -> MnO (4)
g
Number of daily
Mortalitv
Dead/Total
1/20
2/30
6/40
11/30
9/30
3-hr MnO exposures
I"
5
7
15
37*
30
.
. --
fectious challenge.
24 hr after in-
Significant difference when compared
with control mice challenged with
influenza virus .
and congestion. The extent of these changes was greatest in lungs
of mice after the fourth 3-hr exposure to Mn02-
Significantly enhanced mortality rates and reduced relative
mean survival rates (RMSR) were observed following one or more
Mn02 exposures and challenged with airborne K. pneumoniae (Table IV-2
When the interval between the Mn02 exposure and the infectious
challenge was 1 hr increased mortality rates were observed among
mice exposed to manganese for three and four daily 3-hr periods.
A concurrent reduction in mean survival time was also seen.
Increase in interval between exposure to Mn02 and the infectious
challenge to 5 hr resulted in more pronounced increases in mortality
rates and decreases in survival time which were observed after a
single exposure to
Mn02 Exposure and Challenge with Influenza Virus . Mice were
challenged with the -influenza virus 24 and. 48 hr before exposure
to aerosols of Mn02- As seen in Table IV-3 at 24 hr the only signi-
ficant increase in mortality and mean lung lesion scores were
observed following a single 3-hr exposure to Mn02- Repeated ex-
posures to Mn02 also resulted in enhanced death rates but the
statistical significance of the differences could not be ascertained
When the exposure to Mn02 was delayed to 48 hr after the infectious
challenge significant mortality and lung lesions score increases
occurred after both one and two 3-hr exposures to Mn02 . The third
3-hr exposure to Mn02 also resulted in increased mortality rates
and lesion scores but the differences were not significant.
Results of the exploratory study indicate that a 3-hr exposur
to MnO or Mn02 aerosols results in increased susceptibility to
bacterial pneumonia and influenza infection. The increase in sus-
ceptibility was demonstrated by increased mortality rates and de-
creased mean survival time after challenge with airborne K. pneu-
moniae and increased mortality rates and lung lesion scores after
challenge with airborne influenza virus.
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Table IV-3
MORTALITY RATE AMONG MICE CHALLENGED WITH
INFLUENZA VIRUS AND EXPOSED TO Mn02 AEROSOL
Interval Between Virus Challenge
24 hr
Experimental
Condition
Mn02 (l)a
Mn02 (2)
Mn02 (3)
Mn02 (4)
Influenza Virus
Virus
Virus
Virus
Virus
-»Mn02
— Mn02
-» Mn02
-* Mn02
(1)
(2)
(3)
(4)
Mortality
D/T
0/30
0/30
0/30
0/30
6/44
13/34
7/34
9/45
11/47
7.
0
0
0
0
14
38*
21
20
23
Lung
Lesions
0
0
0
0
1
2
1
2
2
.4
.63
.97*
.79
.02
.49
4S hr
Mortality
J3/T
0/5
0/5
0/5
9/30
21/30
19/30
13.30
'*
30
70*
63*
43
Lung
Lesions
2
4
3
3
.57
.03*
.77*
.17
Number of daily 3-hr Mn02 exposures.
Significant difference when compared to control mice challenged
with influenza virus.
Table IV-2
MORTALITY AND RELATIVE MEAN SURVIVAL RATE (RMSR) OF MICE
EXPOSED TO MN02 AEROSOL AND CHALLENGED WITH K. PNEUMONIAE
Interval
Experimental
Mn02 (l)a
Mn02 (2)
Mn02 (3)
Mn02 (4)
K. pneumoniae
Mn02 (1) -> K.
Mn02 (2) -+ K.
Mn02 (3) -> K.
Mn02 (4) -* K.
Condition
pneumoniae
pneumoniae
pneumoniae
pneumoniae
a Number of daily 3-hr Mn02
Mortal]
D/T
0/25
0/25
0/25
0/25
8/31
9/27
5/25
12/25
12/25
K.
L hr
Lty
%
0
0
0
0
26
33
20
48
48
Between
Mn02 Exposure
and
pneumoniae Challenge
RMSR
Days
15.0
15.0
15.0
15.0
13.7
10.6
12.4
9.4*
9.5*
5 hr
Mortality
)/T
0/15
0/15
0/15
0/15
20/61
35/60
36/60
31/60
29/60
%
0
0
0
0
33
58*
60*
52*
48
RMSR
Days
14.0
14.0
14.0
14.0
10.8
7.0*
7.1*
7.9*
8.7*
exposures.
Significant difference when compared to control mice challenged
with K. pneumoniae.
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