High and low barometric pressure effects on susceptibility and resistance to infectio

Gordon, F. B.

English

NASA Technical Reports Server

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Quarterly Statur Report 
1 Jul7 - 30 Saptanbet 1966 
Microfiche (MF) ,A 
ff 653 July 66 
"Bffectr of Eigh and Law Barafaetkic Pressures on 
Surceptibilitf and Reeietan to Infection" 
/ 
Frmcie B. /' Gordon, N.D. 
D$tector, 
Departmane' of Microbiology 
naval Xedic6l Research Inst i tute  
Betherd&, Maryland 20014 
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https://ntrs.nasa.gov/search.jsp?R=19670002921 2020-03-16T16:56:35+00:00Z
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Summary. Durlng the period of t h i r  report t w o  addi t ional  experl- 
mentr on the effect of prolonged incraaeed oxygen tenr ion a t  one 
atmorphere (Pig. 1) on en te r i c  f lo ra  of mice were completed and one - 
' add i t i ona l  and perhaps f i n a l  experiment i n  t h i s  serieo was '  i n i t i a t ed .  
Tentative conclurionr have been drawn. 
f o r  holding mica under increased pressure, up t o  95 pr ig  ( ~ F g e  2 and ' 
3), have permittad i n i t i a t i o n  of a new ae r i e s  of experiments at  t h i r  
prerrure. Final  item8 of equipaent have been obtained t o  allow 
experiment8 a t  rimulated a l t i tudes .  
tianal independent lnvestigatore have been added t o  the  rtaff. 
, ' D r .  Kun-yen Xuang har begun a program of observations on the  produc- 
tioa and e f f e c t  of interferon in vivo and in cell cu l tures  ia v i t r o  
under parabarorir. CAPT Charler E. Myers, DC, USN, L planning 
observationr on the  normal and/or altered f l o r a  of personnel engaged 
i n  experimental diving tertr preliminary t o  Sea Lab I11 or in other  
conf ined rpacor . 
The completion of c h d e r o  
During t h i s  period, two addi- 
Normal en te r i c  bac te r i a l  f l o ra  of mice. 
Experiment $.-The preliminary report of t h i s  experiment war made In 
the Fourth Quartarly S ta tur  Report, 1 Apri l  - 30 June 1966, and the 
f i n a l  r e r u l t r  are now presented in Table 1, and In Figure 4. 
e ~ p e t i m e n t  a l a rge  group of m i c e  were placed together in a olngle  large 
moue box under normal conditionr fo r  three weeks. 
divided i n t o  three groups of 10 mice each, one of which wae kept on 
the rhelf  in t he  utiP.1 room @om A i r  Group), a second group was  placed 
L . i n  8 chaoher through which air fropp a corPpraeeed air tank wa6 allowed 
t o  flow (Tank A i r  Group) and a t h i rd  group (02 Group) war'placed in a 
' r h i l a r  chamber through which a mixture of oxygen, 60% t o t a l ,  In air 
war allowed t o  flow. A l l  three groups were a t  one atmosphere of prearure. 
Culturer of weighed fecal pellatr of each mwoe were made a t  in t e r r a lo  
from QM week previour t o  placing in the  3 e n v i r o ~ l e a t s  t o  6 week. after, 
a8 fadicated Lo Table 1. Although there wera changes La the l eve l  of 
Leveral rpecier  of bacteria during the courre of the e x p e r h n t ,  t he  
t rand was for the  leve l r  in a l l  three of the  groups of mica t o  be 
rhi lar .  Standard e r ro re  were calculated in a l l  caseo. 
In t h i o  
. 
They were then 
Cmpariron of the  l eve l r  of 8 1 0 W  lactore fernrenterr i n  the 3 group0 
, iadicater a r ign i f icant  departure of the 02 group of mice from t h e  two 
con t ro l  groupr. 
a d  by the  charted leve ls  i n  Fig. 4. 
number8 of rlow lactose fermenterr i n  the s too ls  of t he  02 mice were 
considerably l e sa  from those of the 2 control  groups which were not 
r i g n i f i c a n t l y  d i f f e ren t ,  By tho 6th week t h i s  difference had become 
much lerr. Similar differeacer  were seen with an a typica l  (aberrant) 
g. coli ,  which appearr only on plater  Wzwrr rlow lactore f e r m n t e r  
colonies arr prerant  and in proportion t o  them, and f o r  t h i s  roaron 
would ba rxpected t o  follow the curve of t he  latter in Incidence. 
. Thio is Indicated by underlined figureo i n  Table 1 
A t  the  2nd and 4th weekr t he  
. ,'. ' 
, . . .  
. .  . . r  
" (Figures underlined in Table 1). The incidence of a Klebsiella sp. 
was also d i f f e ren t  i n  the 0 s  mice and controls  a t  weeks 4 and 6 ,  
the 02 group having a highe; incidence (Figurer underlined in T8ble 1). 
Experiment S,-The plan of t h i s  experiment was similar t o  t h a t  of 
Experiment 4, except t ha t  an oxygen leve l  of 77% t o t a l  hi air  was used 
instead of the  60% previouely employed. 
received air from 8 tank. 
to 90% total  oxygen in air, but t h i s  l eve l  war found t o  be incompatible 
The control  group of mica 
A160 included was a group of mice exposed I 
i t h  a long term experiment. A l l  m i c e  of t k i 8  last group died batueea 
I I r t  d 4- week etet being placed is this razos~ct , -  Ia 
' cantrut t o  X x p .  4, no rigniffcmt differences were obset*ed b e m e a  
oxygen exposed mice and controls ,  with respect  to slow laetose 
fermenterr and the aberrant c o l i  type. 
behaved rrlmilarly a8 in Exp. 4, and the  r e s u l t s  are depicted i n  
Figure 5 .  
Klebsiella after being placed An the chamber (no recovery f rom 8.1 d 
oxygen group continued t o  show levels of Klebsiella. Theeurva f o r  
the controls, Figure 5, i s  not s ign i f icant ly  d i f f e ren t  than t h a t  f o r  
the oxygen mice, but  the  former includes the negatives (no growth 
from 0.1 m l  of a lom3 di lu t ion ,  i , e . ,  negative at 1.0 m l  x 10-4) which 
are calculated as posi t ive a t  t h e  next lower log d i lu t ion .  
given a pos i t ive  value of 103.4, i.e., 1 of 4 pla tes  poosibly posi t ive 
a t  the  lower d i lu t ion .  
the  difference is probably s ignif icant .  A reasonably s igni f icant  
difference war alro seen between the 77% oxygen group and the controlr 
ix weeks with respect t o  colony type 2 of an anaerobic lacto- - 
bacillurr (Fig, 6 ) .  
oxygen and cont ro l  group6 (t teat) w a s  found to  be less than 0,001. A 
rimilar difference with respect t o  colony type 2 of t h i s  lactobaci l luo 
w u  8een in Exp. 4 at 2 weeks, although the difference was. aot 
The Klebsiella, however, 
' -  
A l l  but one of the control  mice became negative f o r  
crd a 1J-3 dfleEoa)- C!X3X&E:, UZW a d  forit. me Of te Of 
They are 
Since t h i s  confirma the  trend oeen in Exp. 4, 
The P value for the difference between.the 77% 
r t 8 t h t i C a l l y  Signif icilat. 
' Experiment 6 . 4  group of mice w a s  b p t  f o r  a period of 7 weekr in 
roan air and rtool cu l tures  were made a t  0 ,  2 and 7 weeks.. This w a s  
f o r  the purpore of determining whether var ia t ions  in bacterial count 
ruch 88 have been Been i n  cont to lmice  i n  chambers would be oboerved 
in the shelf  mice. ExpecFally between the 2 and 7 week cul tur ings 
the re  war l i t t l e  change i n  level  of the  d i f f e ren t  species. 
mu_ngests t h a t  unrecognized factors  associated with maintenance of 
mice in exporure chamber6 even under normal ai3 at  me atqorphare 
iofluence the  estimation of levels of enteric//bacteria. 
%is 
. 
' '  / 
Experiment 7m-ThL8 experiment, now undeyway, l a  designed t o  test 
I the hypotheeie mentioned above (Exp. 6 ) .  Mice at 77% 02, 'nice in air * .  
, from a coppreesad air tank, and mice i n  drmal room air (ahelf mice) - aro beiw obrerved in the  urual  manner. It lr ant ia ipated t h a t  t h i r  P - -  - 
, . . .  
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Effect  of hyperbaric enviroiunente on suecep t ib i l i t y  of mice t o  _ _  
lreroeol challenge with moure pie 
rteel and p l a s t i c  chambere t h a t  wil .  
and allow t h e  uee of posi t ive prerreures up t o  95 psfg, and skmrhted 
altitude to  uear ly  0 nun Hg, pressure, were f i r s t  used during the 
period of t h i e  report. Sui table  pumps f o r  handling oxygen in the  
hypobaric environownt have recently been acquired. After some practice* ' 
urd t ra ining of pereonnel i n  maintaining mice under preeeure, one 
experiment was colapleted . 
I 
1 
Experiment Mopn 15.-Ten mice (Group A) were held i n  a preeeure 
1 'chamber for 3 weeks at  a simulated depth of 207 ft. eea water (95 prig) 
i n  a mixture of 02 (2.8%) and N2. This providee a p0 equal t o  
rtondard conditiona a t  sea level.  A control  group (Bf were held in. 
the  rame type of chamber in a i r  a t  1 atmoephere. 
mice, avai lable  for challenge at 3 wke, were exposed t o  an aerosol of 
mouse pneumonitis agent (Fig. 7 ) ,  and returned to  t h e i r  respective 
chambers. Two dayr later the  t e a t  mice were euddanly decompressed due 
t o  a leak in the chamber, but  eurvived and were returned t o  prereure 
prersure) died, one only minuter before scheduled sacr i f ice .  A l l  of 
t h e  control  group eurvived but were sacr i f iced  on the 9th day. Data 
on t h i s  experiment are rwxnarized In Table 3. 
grea te r  ruecep t ib i l i t y  of the  pressurized mice, and addi t ional  axperi- 
mente are planned to  determine what fac tor  ac tua l ly  i o  reeponsible. 
One such experiment now underway will compare 3 group8 of m i c e 8  a t  
95 peig with 2.8% 02; at  rimulated 37,000 f t .  a l tgtude with lOoX 02; 
8nd In air  a t  1 atmosphere (0 psig). 
' 
Six t e a t  and 9 cont ro l  
i t h i n  90 minutes. On t h e  9th day a11 6 of t h e b s t  mice (under 
The r e su l t e  point t o  a 
I Effec t  of increased oxygen on Lnrmunizability of mice with Vi antigen 
&e determined by a challenge w i t h  Salmonella ~JA& (Ty 2) . A group of 
2b mice were placed i n  a chamb er wi t IZiYi jZtant  flow ot oxygen ( t o t a l  
77%) in air a t  two l i t e r a  per minute. 
placed In 8 chamber with a i r  from a compressed air  tank flowing a t  the  
6 . ~ ~ 6  rate, and a 3rd group of equal s i ze  were kept; on a shelf in  the  
room. After  7 days in theee environments the mice of each group were 
arranged in to  4trubgrwpo of 5 mice each, and injected subcutaneously 
into the  neck region with a ringle dose of V i  antigen in tenfold vary- 
ing amountr no indicated in Table 4. They were then returned t o  t h e i r  
respect ive environmentr. 
liter (0.25 al) of Salmonella typhi (Ty 2) . Thie represents approxi- 
u t a l y  5 i p  LD5080. Three days a f t e r  iuoculatian when the  e f f e c t  of 
tho challenge could bo expected t o  be complete the  number of dead and 
A 2nd group of 20 mice were 
Six daya later the  mice we e challenged 
' ln t raper i tonea l ly  with a sa l ine  euspenaion of 6 x 10 5 ce l l a  per m i l l i -  
I 
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Studier  on a l t e red  gaseous environment a t  the cellular level.  (Thlr 
por t ian  of the report prepared by Doctors Neptune and Weise). As I 
I 'noted in the  last repor t ,  a l t e r a t i o n s  occur h microbial enzymatic t ! 
*l I 
/ 
Aa reported before, t h e  pyrtavic oxidase of meningopneumonitis agent 
(Chlamydia) i 8  rt imulated by oxygen. 
hypothesir t h a t  inh ib i t ion  of pyruvic,/oxidase i e  one of the epec i f lc  
It ha6 been reported 
t h a t  hydrogen peroxide raac te  a p o n p e o u e l y  with pyruvate "d the  
pyruvate ir decarboxylated, 
laboratory in conalderable de tu i l ,  
any doubt E202 and pyruvate do ,form an highly unstable mixed anhydride 
.whlch deca~pores  to  yield C%,/wttter and acetate. As reported before, 
t h i a  microbe ham a pentose phosphate pathway which we have portrayed 
in Fig. 8. Note t h a t  the terminal aequence on the  r i g h t  involver a 
per0~ id8 t i01k  reac t ion  w i t h  the  generation of H202. I f  we asourPo that 
endogenow t r a f f i c  over t h i s  pathway is continuouo and t h a t  endogenoua 
H202ir decarboxylating pyruvata, then destroying pa r t  o r  a l l  €1202 
rhould decrease the amount of C02 re leased f r a n  pyruvate. 
war r tudied In the  presence of added catalase there  was d e f i n i t e l y  
le06 r t l r u l a t i o n  by oxygen a8 shown in Table 5. 
Since the  endogenous metabolism t h a t  produces H202 Le presumably 
not maxlmal, it reemed important t o  attempt maximal etimuletion and 
: obrerve the decarborylation of pyruvate, The obvioue experiment Fa 
f t o  add excess glucos0-6-Plj~JJADP and GSSG. 
enterr not only the  pentose phhaphate pathway but  is a l s o  degraded 
t o  pyruvate. 
f p d  pyruvate with added radioactive pyruvate and thus the y ie ld  of 
C % f r a n  pyruvate appears t o  be diminished, Therefore, it was 
nacerrary t o  add an inh ib i tor  t h a t  would impede the  breakdown of  
This finding l o  contrary t o  our 
' 
-=chanisnu fo r  the  toxic e f f e c t s  of,oxygen. 
I 
. Tliie Meaction has been s tudied in our 
The r e e u l t s  ind ica te  t h a t  without 
' 
. 
The enzyme 
I -  catalare destroyr E&. 
. 
When the pyruvic oxidase of meningopneumonitir 
\ 
. 
Glucose-6-PO4 , however , 
Thlr latter r e a c t i o n p e r n i t s  the  mixing of  metabolica11y 
r 
- glucora-6-PO t o  pyruvate. k - c h o r e  iodoacetata which i n h i b i t r  ! 
tlycctaldeh$e-3-P01,-lW~ dehydrogenase , I 
mcdertly inhibited by iodoacetrte, whereas the pathway t o  pyruvat. 
Therefor., 
1 
Aa rhom in T8ble 6 t he  pentose pathway (carbon 1) l a  only i , 
6 (carbon 3 and 4) ir reveraly deprerred by iodoacetate. 
,sit appeared t h a t  experlmentr w i t h  iodoacetate ware fear ible .  
* The data  in Table 7 c l e a r l y  indicate tha t  in the presence of 
I ' glucor.-6-P04 there  i r  8 marked r t imu la t im  of the  decarboxylation 
of pyruwate and t h a t  t h i s  st imulation i r  abolished by catalara. 
. A  f a c i l i t y  har been ret vp, including a laboratory, equipment and 
I 
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I I 
I 'I ' ! 
1 Effect of parabaric conditione on induction and e f f e c t  of intarfaron. 
{I - 
reagentr f a  p e r f d u g  quant i ta t ive tests fo r  interferon. Standard 
i n t e r f e r m  inducer# urd interfaron prapara t iow have been prepared urd , 
. I  
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Environment i n  which mice were kept. (1 atmomhere) 
Tank air 
I 
I 1 
Slaw lactose -1 8.98 8.64 8.08 I 
1 
4 7.28 - 6.86 5.75 i 
4 
2 - 8.11 7.73 6.25 I ,  
i l  
. . e  . fermenters 0 8.52 8.02 8.49 I , * I  
I * ,  
1 7.57 8.13 7.36 
5 r80 
. . 4  6 '  6.19 6.75 
!I 
4.49 4.76 -5.05 :I I,
3 -71 3.95 4.30 1, 
3.80 . 4.02 4.38 1 I 
ji 
! 
3.65 
3.56 LZ! 
t ,  
&bs i e l l  a 5.28 5.35 5 .44  
2* 
' r  (3.40 .63 
4.12 
'! . .  
7.80 . 7.66 8.24 1 
1; 
7.06 .  8.16 7.61 \ 
c 
8.64 8.78 7.75 
8 . 3 4 .  8.29 8.03 I 
8.41 ' 8.33 7.80 " 
I 
g 8.01. 7.96 8.13 
I 
-1 7-71 7.53 
0 8.61 8.62 
1 8.27 8.10 
2 7.02 
6 7.86 8.85 
1 
. l  4 8* w 
i 
7.91 I 
8.46 
5.8s 
7.28 
7.81 
7.89 i 
\ 
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1 
1 \ '  
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(Table continued on next page) 
. I  
Table 1 (continued) 
Bacteroides 
colony type 1 
colony 
. .  . 
-1 
0 
1 
2 '  
4 
6 
t y p e 2  -1 
0 ,  
1 
? '  6 
. 49. 1- ! ..- . . . I  
8.07 I 
8.48 
6.92 , 
7.66 , 
7.82 
8.99 
8.81 
8.63 
6.19 , 
7.62 ' 
7.93 , 
6.76 
7.97 
8.30 
7.92 
7-24 
8.22 
8.94 
8.67 
8.99 
6.79 
7.39 
7.91 
6.52 
8.28 
8.04 
7.39 
7.33 
7.64 
8.93 
8.78 
8.68 
6.76 
7.72 
7.40 
I .  Table 2. (Exp. 5) 
Numbers of bacter ia  per gram. 
metric means of counts from 10 mice). 
Stool cul tures  of mice i n  3 type6 of gaseous environment. 
(Figures are logarithms of t he  geo- 
I 
I 
. .. ~ I . .  - .  Environment i n  which mice were kerb (1 atmosDhere) 8 
’ .  Type of I n t e r v a l  < 
bacterium . (weeks) .Tank air  02(90$ t o t a l  i n  a i r )  02(77% t o t a l  i n  air) i 
I > I  
1 E. c o l i  -2 8.91 
I Taberr ant ) 0 3.66 
+2 7.74 
+4 5.67 
+6 . 5 -79 
I .  
1 
8.57 
3.76 
6.02 ,-* _- 
I ,  
6.92 \ 
3.81 I’ 
‘ f  
! 
ti 
7.31 
5.05 
5.68 
8.35 
0 8.80 
anaerobio, I ”  -+2 . 6.68 
n y t y p e l  +A 3.76 
6.19 ‘ 
7.95 
8.85 
8.18 
6.95 
x i 2  I ’  
8 a, 8.77 
1 8.92 
8.75 
-- 
i’, .. - i .  . ,l 
’ I’ I# 8.82 8.44 8.64 * 
9.112 9.30 9.45 4 
7.26 8.28 7.26 
8.76 -- 8.58 
4.18 -- 5.46 
I 
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k 
n next page) i k i n  text  a re  underlined. 
between 2nd and 4th week under a 90% oxygen atmosphere. 
+ 
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.A ? 
e .  Table 3.-  Effect of pressure on s u s c e p t i b i l i t y  of mice t o  ' 
. . -._ ). . 8 I. .-, plousc pneumonitis agent .(
A. Mice held a t  95 psig 
i n  2 . w  O2 i n  NZ. 
B. 
- 
Mice held i n  air  
a t  0 psigi 
; i 
I .  
i/T t 5/b 
Grooa changes i n  lung, 2*8*  
2.1' 
1 
ree of i n fec t ion  
2.0 
8.6 x lo5 (1 s a c r i f ' d  
mouse) 
er of agent i n  lungs 
' 8  
. . .  .. 
. .  . .  
, _  ... 
. .  
4.1 105, ( 9  
s a c r i f t  d mice) 
. .  
4.2 x 105 ( 5  mice 
fuund dead) 
. .  
I 
2. . 
v .. , 
=,-. - 
;,At\ .,. .,*$A I * \> " 
Table 4 . -  Resistance t o  challenge of mice immunized t h  V i  ant igen w i  
after being held i n  77% O2 and i n  air. 
4 
Result of challenge with sal. typhosa; 
dead/total mice 
Tank a i r  Room a i r  
3/5 4 / 5  
Table 5.-- Pyruvic Oxidase of men,ngopneumonitis agent; 
response t o  catalase - .  , " .  
I / 
Addition Gas Phase 
.I 
02 
41 48 
37 43 41 
Air N2 
None 36* 
Catalase 
*qmoles CO2 
Each flask Contained a high Kt, low Na', phosphate 
buffered solution and 0.4 pc and 4 pmoles of pyruvate. 
300 units of ster3J.o catalase was added t o  appropriate 
flasks,  The f ina l  volume was 1.9 ml and the pH 7.0. 
The f lasks were incubated f o r  2 hours a t  34 C .  
0 '  
1 
+ 
L 
, .  
, . .  
1.1 , . 
Table 7.-- F;yruvic oxidase of MN agent 
Addition q m o l e s  COz/mg prote in  
None 66 
54 I odoacetate 
4 
G-6-P, NADP, CSSG 
G-6-P, NADP, CSSG, I odoacetate 12 0 
32 G-6-P, NAIIP, CSSG, Iodoacetate, Catalase 
As indicated,  flasks contained 4 kmoles of pyruvate 
, (I-CU), 2 paoles Tf glucose-6-P04, 3 pmoles of N U P ,  
&, was 300 fimolar units .  
10 p o l e s  of NADP. 
cohditions were a3 noted i n  Table 6 ,  
Iadoacetate was 10-3 M and ca ta lase  
The gas phase was air. Other 
. .  
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holding mice at: various atmoepheric pterrurcr from 
altitude) to 95 p i i g  (eimulated depth of 207 ft in 
varying presrurer of 02, N2 a d  air. 
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Opporfte end of 
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LACTOSE FERMENTERS;STOOL 
A .  ROOM AIR i 
I 
AIR FROMTANK 
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- I  0 + I  2 4 6 
WEEKS 
Chert of ievelr of slow lactose 
in Experiment 4, tcvels of the 
pattern. ( I  
fermenterr in the 3 groapr of mice 
atypical E-. were of the #.me 
I -. 
L 
. .  
I 
; 4'. 
I 
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KLERSIELLA ; STOOL 
1 :  I I I I 1 I 
- 2  0 + 2  + 4  + 6  
WEEKS 
- - - __r__ - - --(-I - 1  " tm- //-I.- _-  _- 
hart of lrvclo of Kltbslella d e b  in the mice of gxpariasnt S. 
etected It each cul alro indicated. # ,  YL,- 
-- 
Murborr of mfce, of the total of 10, in which th io  bacterium ~ a i  
I ,  a 
-3. 
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mal 1 
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PENTOSE PHOSPHATE PATHWAY 
-2- $ 
PCU v.29 I GSSG 
Reductase Peroxidase 
G-6-P 
6-P-G 
Dehydrogenase 
.. . 
. .  
i: 
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ursd by menlngopneuaOnltid agent. 
. . .  . .  


Effects of High and Low Barometric Pressures on Susceptibility and Resistance to Infection Quarterly Status Report, 1 Jul. - 30 Sep. 1966

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