Effects of staphylococcal enterotoxin B on functional and biochemical changes of the lung in rhesus monkeys

Toxiroa, Vol. 16, pp. 343-330. ^,ç Perpmon Pros Ltd. 1978. Printed in Crreat rritnin .

0041-0101178/1101-0343f02.00/0

EFFECTS OF STAPHYLOCOCCAL ENTEROTOXIN B ON FUNCTIONAL AND BIOCHEMICAL CHANGES OF THE LUNG IN RHESUS MONKEYS* C. T. Lnr, R. D. DeLAUrER, M. J . Gwr'FIN and C. L. HADICIc U.S . Army Medical Research Institute of Infectious Diseases, Fort Derrick, Frederick, Maryland 21701, U.S.A . (Acceptedjor publication 13 March 1978) C. T. Ltv, R. D. DEI,wrEa, M. J. GRIFFIN and C. L. H~ntctc. Efforts of staphylococcal cnterotoxin B on functional and biochemical changes of the lung in rhesus monkeys. Toxicon 16, 543-SSO, 1978 .-Ef%cts of ataphylaeo~cal entaotoxin B were studied in aneathetimd rhesus monkeys. During the period from 6 to 11 hr following i.v. igjection of the toxin (1 "0 mg/kg), respiratory quotient increased, while functional t+esidual capacity, OOi output, O= oo»sumption and expirod COs concentration da~esaed . By 11 "5 hr, the surface tension of lung extracts and total lung water content increased, as shown by simultaneous accumulations of extratxllular Na+ and water. These results provide evidence to support a hypothesis that pulmonary dysfunction and terminal pulmonary edema contribute to dealt during enterotoxemia by staphylococcal B in rhesus monkeys. INTRODUCTION

Several staphylococcal enterotoxin B-induced physiologic changes, including peripheral capillary pooling (B>r1seL, 1972), intracellular dehydration, hypovolemia (Ltv et al., 1976b) and decreased cardiac functions (Lru et al., 1977) have been considered as possible causes of death during such toxemia in rhesus monkeys, the exact mechanism for induction of irnversible shock during enterotoxemia is still unknown. Pathologic studies revealed that pulmonary interstitial edema and increased lung weights were associated with i.v. inoculation of staphylococcal eaterotoxin B in monkeys (FI1vlcoi.D, 1%7 ; íSTILES and DeNrvISroN, 1971). This investigation was undertaken to study changes induced by this enterotoxin on pulmonary function, metabolic rate and arterial blood gas tensions, as well as lung water content, distribution of electrolytes, and surface tension in anesthetized monkeys. ALTHOUGH

MATERIALS AND METHODS

Healthy male rhesus monkeys (Maaaca »wlatta) wroighing 3ß-S"9 kg were allocated into control (N~9) and toxin-inoculated groups (N6~. The latter group of monkeys had no detectable serum antibody against staphylaooocal enterotoxin B prior to their use. Approximately ~ hr before the expa~imemt, urtilstalal polyethylene catheters (PE 190, i.d.~1"19 mm, o.d.~1"70 mm) were placed in the femoral artery and vein "In conducting the research described in this report, the investigators adhered to the "Guide for the Caro and Use of Laboratory Animals," as promulgated by the Committx on the Revision of the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Raoaroea, National Research Council . The facilities are fully accredited by the .4rlaícan Association for Accreditation of Laboratory Animal Care. The views of the authors do not purport to reflect the positions of the Department of the Army or the Department of Defense. This work was reported in part at the Fall Mating of the American Physiological Society, Philadelphia, Pa., August, 1976 [Physiologist, Washington 19, 273 (197] . 543

344

C. T. LIU, R. D. DELAUTER, M. J. GRIFFiN and C. L. HADICK

under Ketamine anesthesia. Monkeys were restrained in primate chairs for recovery from anesthesia (Ltu et al ., 1977)" Highly purified staphylococcal enterotoxin H ( >99 ~) (ScxArrrz et al., 1965) at a single dose of 1 mg/kg was injected into the femoral vein of experimental monkeys, while controls received only isotonic saline. Measured values were compared between normal and toxin~hallenged monkeys under identical experimental conditions . All monkeys were sedated with Ketamine (13 mg/Itg, i.m .) and placed in a supine position approximately 3"3 hr after i.v. injection of staphylococcal enterotoxin B. Endotracheal and intraesophageal intubations were completed within 30 min. Various pulmonary functions, metabolic changes, and blood pH and gas tensions were determined hourly betweat 6 and 11 hr after toxin or saline injection . However, one additional ezperituental monkey died 7"3 hr after enterotoxin inoculation ; its data are reported separately. Detailed information on txhniques for measuring respiratory and metabolic variables in anesthetized rhesus monkeys were described previously (Lrv and DeLAVreR, 1977). The basis of using such methods was that relatively constant respiratory and metabolic functions could be obtained for a period of 6 hr in lightly anesthetized normal monkeys. The voltune of Ox consumption was corrected for temperature, pressure and water vapor saturation . Arterial bloodpH, PO,, PCO,, HCO,' , total CO= and base excess were determined on a blood gas analyzer (Model 163, Conning Scirntific Instrtuneats, Medfield, Mass .) . Within l5 min after completion of pulmonary studies, various tissue samples including a lobe of the right lung, were excised from the anesthetized monkey. Lung samples were immediately blotted with absorbent paper to remove surface blood and for determinations of surface tension, water contrnt, total lipids, phospholipid and electrolytes p> including Na+, K+ (.OvsRtNArt and DAMS, 1947), Cl' (CoTt,ove et al., 1958) and phosphate phosphorus (Fts~ and SUHBAROW, 1925). Lung tissue was homogenized in distilled water (1 :10, w/v), and surface tension of the supernatant measured with a tensiometer (Model 21, Fisher Scientiftc Co., Pittsburgh, Prnn .) . Water content was determined by drying approximately 0"2 g of lung tissue to a rnnstant weight in an oven at 110°C. The Cl- content in the dry minced specimen was extracted for 20-24 hr in 3 ml of distilled water, while Nat, K+ and phosphate phosphorus were extracted from the homogeniud wet lung tissue with S ml of LO'á trichloroacetic acid . Distributions of intracellular and extracellular water and electrolytes in the lung were calculated according to the method of Bertsort et al., (1956) . Tissue phosphate extract was determined as total acid-soluble and inorganic acid-soluble fractions. The former was prepared using concentrated HaSOa digestion at 3í0°C and the latter proceeded without acid digestion . Organic acid-soluble phosphate was calculated from the difference between total and inorganic acid soluble phosphorus . Total lipids in the lung were extracted with a chloroform-methanol mixture (2 :1 v/v) for 20-24 hr at room temperature. The weight of total lipids in the lung was measured gravimetrically after complete evaporation of the solvent from the purified extract in a vacuum oven. Phosphorus contrnt of phospholipid was determined by the method of BARTr.Err (1959) . The phospholipid concentration was obtained by multiplying the phosphorus value by a factor of 25 . Values obtained from the experiments were compared using analysis of variance with repeated measurements. The values at 6 hr were used as the base litres for each monkey to test for significant changes over time. Further, differences from base lines for the control and enterotoxin-inoculated groups were also compared by an independrnt t-test . The Null hypothesis was rejected at the S"ó level.

Pulmonary functions and blood gases

RESULTS

Effects of i.v. inoculation of staphylococcal enterotoxin B on tidal volume, pulmonary ventilation and pulmonary mechanics in anesthetized rhesus monkeys breathing room air for (1 hr are summarized in Table 1 . No significant changes were found ; however, dynamic pulmonary resistance, functional residual capacity and expired COz concentration decreased significantly as compared to control monkeys in the later period (8-ll hr) after toxin injection (Fig. l). The respiratory rate was maintained relatively constant as compared with controls, in which decreased values were observed as a possible result of continuous anesthesia (Fig. 2). Further, one monkey that died earlier (at 7~5 hr) showed increased respiratory rate, dynamic pulmonary resistance and arterial blood PCOz while its tidal volume, dynamic pulmonary compliance, intraesophageal pressure, expired air flow and arterial P02 decreased markedly from control values (Table 2). No change in blood pH was demonstrated (Table 2). As compared to controls, rhesus monkeys showed significantly decreased O~ consumption and increased respiratory quotient values at 6-8 hr after toxin inoculation (Fig. 3). However, the magnitude of changes in Oz consumption and respiratory quotient remained unaltered

SEB and Respiration in

Monkeys

343

TADLE I . EPPECrs OF STAPHYLOCOCCAL ENTEROTOXIN B (SEB) ON TIDALVOLUK, PULMONARY VENTILATION AND PULMONARY MECHAMCd nV AIV~rüETIZED RHióUB MOAi1~Y8 DREATHINO ROOM AIR COI~ARED i0 ODNTROLS Variable

Tidal volume (ml) Minute volume (L/min) Intraesophageal pressure (cm H_O) Transpneumotach pressurc(cmH~O) Transpuhnonary pressure (cm HSO) Expiratory flow (ml/sec) Inspiratory flow (ml/sec) Dynamic pulmonary compliance (ml/cm H,O) Specific compliance (compliance/FRC x 10~ ~) 'Controls,

N=9;

9

SEB,

~

Values by hr post-SEH (mean f S.E.) 6 7 9 11 3Sí4 36f4 41 f6 36íS 38f 10 38f 7 32 íS l "36í0" 16 O"98í0"13 l "44í0"26 l "25í0"17 1 " 16í0A8 l"28í0"17 l "07í0"14 -S"7 f0"6 -S"6 f0"8 -7"1 f0"9 -4"78f 0"9 -S"59f 1 "4 -S"42 f 1"S -6"40f1 "8 1 "13 f0"09 0"85 f0"07 1 "OS íO"12 O"93í0"10 O"91í0" 16 O"98í0"12 O"90í0"09 ~"54f0"SS --4"7Sí0" 78 -6"O3í0"91 -3"85f0"85 -4"tí9 f 1 "22 --4"44f1 "42 -530f 1 "69 66"6 ítí"1 56"9 f8"7 61 "9 í7"S 60"6 f 10 59"6f 11 66"2 f 11 61 "8 f10 64"0 f5" 1 48"4 f3"7 59"S f6"8 52"8 f6 Sl " 3f9 SS"6 f7 SI "1 f3 8~2 f0"9 9"4 f1 "8 7"9 íl "7 ß" 6 f 1 "7 7" 1 f 1"1 9"1 f1 "9 6"8 í 1 "S 72f9 83f 14 S2í 7 77f 18 71 f 13 101 f22 113 f48

Group' Control SEB Control SEB Control SEB Control

S®

Control SEB Control SEB Control SEB Control SEB Control SEH

N=6.

r 0

6

1

~!

q

II

HOUIIS

fla . 1.

EFPECT oF rNTRAVENOVa srAPHYLOOOCCAL ENTmoTOxtx H (SEH) (1 ma/ka) oN DYNAMIC PULMONARY RF~.TrANCE, PUNCI70NAL R~UAL CAPACITY AND EXPIR® OOs TN AiVESrHETIZED RHEdUS MONICEY3 .

as a function of time between 6 and 11 hr after staphylococcal enterotozin B inoculation. Although C02 output and expired CO s concentration were signiScantly decreased at 11 and 9-11 hr, respectively, after toxin inoculation as compared with the control group of monkeys

C. T. LIU, R. D. DELAUTER, M. J. GRIFFIN and C. L. HADICK

S46

6 viW

U Y U

a

HOURS FB3.

2. EPFECT OF INTRAYENIWS STAPÜYLOCOCCAL ENTEAOTOXIN B (SEB) RESPIRATORY RATE IN ANESTHETIZED RHESUS MONKEYS .

(I mg/kg)

ON

TABLE 2. CHANGES IN PULMONARY FUNCTIONS AND ARTERIAL BLOOD pH, PO3 AND PCO, DURING rmTnmvAt srAPHYLOCOCCAL ENTFaoTOxIN B (SEB) ENTEROroxEMu (O'S br IIEFORE DBATH) OOMPARED TO OONTROLS

ValUe3 (meanfS.E .) Control SEB

Variable Tidal volume (IM Raapitatory rata (cycle/min) Dynamic pulmonary compliance (mllcm HBO) Dynamic pulmonary resistance (cm H~O/liter/sec) Intraesopha,eal pressure (cm H=O) Expired airflow (ml/sec) pH Artanal Blood POs (mm Hg) PCO= (mm Hg) fn i 201

Fra. 3.

OXYOI?N

38 f 13 33 f S 8~3=4~3 31 ~-7

19 57 1 ~8 168

~~3~0~7 62 i2 7~375~0~008 88~6 f4~9 29-0 f 2~7

-IS~7 47 7S18 33~3 38~8

oxwcn ca~giwrT~aa

O CONT11p~,f lN.f1 " fCf ~N ." 1 ~ ~spOf

EFFECT O! INTRAVENOUS SiAPHYL000CCAL BNTEROTOXIN B (SEB) (1 mg/1cg) oN COI~tJMI"rFON AND RE4IRATORY QUOTIENT IN ANESTHETIZED RHESUS MONKEYS .

SEB and Respiration in Monkeys

2 ro E w i 1 a

O

z"ce i i

34 7

eoz ounuT

I

_2

_ , .J i

0

6

7

B HOURS

9

i0

~I

FIG. 4. EFFECT OF INTRAVENOUS STAPHYLOCOCCAL ENTEROTOXIN B (SEB) (I mg/kg) ON COZ OUTPUT AND EXPIRED COz DIJRING Oz BREATHING IN ANFSrHETIZED RHESUS MONKEYS.

TABLE

3.

Variable pH

EFFECTS OF STAPHYLOCOCCAL ENTEROTOXIN B (SEB) ON ARTERLAL BLOOD pH, GAS 7ENSION, TOTAL COz AND BASE EXCF~S IN ANPárHETIZED RHESUS MONKEYS BREATHING PURE Oz Group'

Control SEB PO~ (mm Hg) Control SEB Control PCOz (mm Hg) SEB HCO,' (mmole/liter) Control SEB Total CO, (mmole/liter) Control SEB Base excess (mmole/liter) Control SEB 'Controls, N~9, SEB, N=6.

Values by hr post-SEB (IneaIt~S.E .) 6 7 9 11 7974f0"009 7"362f0"008 7"365f0"Ol 7"373f0-013 7"394f0"020 7"383f0-017 7"378f0"021 367f32 389f23 411 f 16 413f17 413f24 392f17 399±30 33"4 ~2 "4 33-0 f2 "8 31~3 f2 "6 34"2 f3 "3 33 "3 -_~-2~7 32" 1 y3-0 29 "2 ~3~3 19" 1 f 1 "S 19-0 -~ 1 " 7 18 " 1 -~ 1 ~7 18 "1 f2-0 18 "9 ~-1~9 16"4 f1~7 13"9 -2 "2 ZO"4 =1 ~3 20"6 +_ 1 "7 19"6 == I ~6 21 ~3 ± 13 21 "9 ~ 1 "3 20"3 f l "6 19"0 ~2~5 -3 "7 f 1 "3 ~"2 f 1 "4 -3-0 f 1 "4 ~"7 fl "2 -3 "9 a-1~3 -S"9 fl "2 -6"S -~2"0

(Fig. 4), arterial blood pH, PO=, PC02, HCO, -, total C02 andbaseexcess showed little or no changes during the entire I 1-hr experimental period (Table 3). Lung surface tension aiul biochtmical changes Although plasma water content and electrolytes concentrations were not altered after inoculation of staphylococcal enterotoxin B in anesthetized rhesus monkeys ('Table 4), surface tension, organic acid-soluble phosphorus, total water content, extracellular Na+ (mF.q/kg fat-free wet tissue) and total C!- (mEq/fat frce dry tissue) of the lung increased significantly as compared with lungs from control monkeys (Tables 4 and 5). There was also an apparent shift: of intracellular lung water and Na+ to the extracellular space in toxinchallenged monkeys (Table 4). No significant changes were observed in K+ distribution, total lipids, phospholipids and total and inorganic acid-soluble phosphorus of lungs 11-12 hr after a lethal i.v. dose of staphylococcal enterotoxin B ('Tables 4 and 5).

348

C . T . LIU, R . D . DELAUTER, M. J . GRIFFIN and C. L . HADICK

TABLE 4. CHANGES IN WATER AND ELECTROLYTE IN PtASMA AND THE LUNG OF.STAPHYL000CCAL Ei~'rEROTOXFN B (SEBFINOCULATED RHFSUS MONKEYS Sample Plasma

Variable

H=O (g/dl) NaT (mEq/liter) K- (mEq/liter) CI - (mEq/liter) Lung Total Hx0 (g/kg FFWT') Extracellular H,O (g/kg FFWT) Intracellular H,O (g/kg FFWT) Total Na (mEq/kg FFW'i~ Total Na (m1Eq/kg FFDTt) Intracellular Na (mlF.q/kg intracellular HBO) Extracellular Na (mEq/kg FFW'1~ Total K (mEq/kg FFWT) Total K (mEq/kg FFDT) Intracellular K (mEq/kg intracellular H:O) Extraoellular K (mEq/kg FFW'ï) Total Cl (mEq/kg FFWi) Total CI (mEglkg FFD7) 'FFWT=fat free wet tissue. tFFDT=fat free dry tissue . $N= 3 . 4N~6. ilBy independent t-tat, P <0"O5 . !;Hy independent t-tat, P <0" 01 .

Values (mean Control (N=4) 93 " 0 _-_0 143-2 3~57y022 113 y4 807=8 386=-33$ 42l =60$ 819 -_2~6 429-=26 62 " 8 -3~0$ 46 " 5 _-_3'0$ 633 143 326=21 161116 1 " 46 .=0"27 548 157 290 ~ 3 l

.-S .E .) SEB (N=7) 93 _ 0~ I 143 _:~ 3 3 " 65 -=-0 " 19 108-4 R41 -- 9 512=314 329 ~ 36§ 76~3 -= -5~8 457 _- 44 34~8 - 9~14 66~6 -_3" 14' 53~0 5~7 31 I =_ 38 162 - 22 1 . 80__p . I7 642 : :2 " 74 3891164 .

TABLE S . CHANGES IN SURFACE TENSION, TOTAL ACID-SOLUBLE PHOSPHORUS (TAS), rNORGANIC ACID-áOLUBLE PHOSPHORUS (IAS), ORGANIC ACID-SOLUBLE PHOSPHORUS (OAS), TOTAL LIPIDS AND PHOSPHOLIPID3 IN THE LUNGS OF CANTROL AND STAPFIYLt.)000CAL ENTER01'OX1N B (SEB}R~OCULATED MONKEYS Variable Surface tension " (dyne/cm) TAS Immole/kg FFV1rI'+> IAS (mmok/kg FFW'T) OAS Immole/kg FFWT) Total lipids (g/kg) Phospholipids íg/100 g) 'Control, N=3, SEB, N=7 . tBy independent t-test, P <0-0S . $Fat-free wet tissue .

ValUeS (mean-=3 . E .) Control (N=4) SEB (N =6) 42" 70-!- 095 52~07~= 202+ 1029 110"4 1322 -.=11 ~2 50" 3 1 9 " 9 40" 7 -- 4~0 52 " 6 f 7~4 93 " 6 ~ 12~6t 2136-!- 3 " 76 15271 1 ~73 180= 0" 27 148 _- 0~1 I

DISCUSSION Since few changes in pulmonary functions were observed within S hr after i .v . injection of staphylococcal enterotoxin B in anesthetized rhesus monkeys, the present study encompassed the period 6-I 1 hr after toxin inoculation . During this time, cardiovascular and hepatic functions are severely depressed (Lttr et al., 1977) and body fluid volumes are significantly decreased (Llu et al., 19766) . Since toxin-challenged monkeys may survive for a few more hr beyond the I 1-hr experimental period, the present data only reflects change at approximately the mid-point of staphylococcal B toxemia. According to our limited observations . a toxin-inoculated monkey under anesthesia showed some important terminal changes including hypoxia, hypercapnia, decreased dynamic pulmonary compliance, and increased

SF.B and Respiration in Monkeys

349

dynamic pulmonary resistance. The reason for presenting data from only one monkey was that all toxin-inoculated monkeys (N=6) survived during the 11-hr experimental period and this was the only monkey that died (7 "5 hr after the injection) . During the period of staphylococcal B enterotoxemia, decreased functional residual capacity values were obtained . The possible cause for these decreased values, without a simultaneous increase in tidal volume may be due to an accumulation of fluid in the lung or partial collapse of the lung. The former assumption is supported by the increased lung water content, and the latter is evidenced by the increased surface tension of the lung extract at 11 hr after i.v . SEB inoculation . Since lung-surfactant is responsible for preventing lung collapse through the mechanism of decreased pulmonary surface tension (GREENFIELD, 1974), an increase in lung surface tension may also indicate that lung-surfactant concentrations were decreased in toxin-challenged monkeys. Metabolically, both total-body Oz consumption and COz output decreased, while respiratory quotient values increased to more than 1"0. These findings indicate that (1) cellular metabolism was depressed, although fever might be present (BetsEt ., 1972 ; CRAW7.EY et a1., 1966), (2) anaerobic oxidation was involved, (3) carbohydrate (glucose) was the main fuel for energy release and utilization and (4) a steady state was not achieved . Various forms of phosphorus in the lung, including phospholipid and inorganic and organic acid-soluble phosphates, were measured in control and toxin-inoculated monkeys. Among the three categories of phosphorus, only organic acid-soluble phosphate showed a significant increase after toxin inoculation . Since organic acid-soluble phosphate is a mixture of creatine phosphate, ATP, ADP, and AMP, it is unknown which substances) is(arc) affected . Further, the significance of increased organic acid-soluble phosphorus in the lung during staphylococcal B enterotoxemia remains unknown. Staphylococcal enterotoxin B-induced pulmonary edema, as has been shown by others, is characterized by increased lung weights and pathological changes of pulmonary capillary endothelial cells (F~NECOLD, 1967 ; STtI.ES and DENNISTOIV, 1971). No data were reported on lung surface tension and distribution of intra- and extracellular water and electrolytes . Results from the present study confirm the presence of terminal pulmonary edema in monkeys following a lethal i.v . dose of enterotoxin B. This is based on the finding that total water content, extracellular water and Na* in lung tissue wen increased at 11 hr after i .v . injection of toxin. When lungs are filled with water to a critical level, pulmonary exchanges for Oz and COz are impaired and oxygenation of vital organs is dxrcasod, leading to death within 20 hr . Such rapid death minimizes the likelihood that renal, hepatic, or gastrointestinal changes are directly involved in the lethal eûect of i.v. staphylococcal cnterotoxin B. The primary mode of action of staphylococcal enterotoxin B is known to involve vomiting and diarrhea after ingestion in man and rhesus monkeys. Mxhanisms for such toxininduced gastrointestinal disturbances have been reported from this laboratory (Et.wtiz.t. et al., 1975 ; Ltu and DUFAULT, 1977). The effect presented in this paper on lung tissues is probably only secondary, obtained in monkeys intoxicated by the parcnteral route at an extremely high dose of staphylococcal enterotoxin B (1 mg/kg) . At this unphysiological dose level, cardiac, renal and hepatic toxicities have also been shown in the rhesus monkey (Liu et aJ., 1977 ; Ltu et al., 1978). Results from our preliminary studies reveal that continuous positive-pressure breathing, maintained at 3~ cm Hz0 for 2 days, may prevent death in enterotoxin B-challenged monkeys (Ltu et aJ., 19?6a) . These findings indicate that continous positive-pressure breathing is effective in preventing toxin-induced pulmonary edema, despite further

SSO

C, T. LIU, R. D. DELAUTER, M. J. GRIFFIN and C. L, HADICK

decreases in cardiac output and blood pressure (KIRBY et al., 1975). Because staphylococcal enterotoxin B-challenged animals continue to survive after 2 days of treatment witli continous positive-pressure breathing, it is suggested that pulmonary capillary membranes are not critically damaged, or that capillary damage is reversible within a relatively short time. It is concluded that respiratory dysfunction and pulmonary edema are major contributing factors leading to death of rhesus monkeys after i.v. staphylococcal enterotoxin B-inoculation. Acknowledgements-The authors thank Doues R. DuF~tn.r and Gt,stvx E. CRONE for technical assistance, Gt.Ety A. Htßst:E for statistical analyses, PtreaE W. $UMMERa for editorial assistance and RECttv~ E. Sr~t-EY for saretarial aid. REFERENCES B~ttrt.Er-r, G. R. (1939) Phosphorus away in column chromatography. J. blot . Chem . 234, 466 . BEtsra., W. R. (1972) Pathophysiology of staphylococcal rnterotoxin, type B, (SEB) toxemia after i.v. administration to monkeys . Toxtc+rn 10, 433. BENSON, E. S., FR EtER, E. F., H~t.uWaY, B. E. and Jotrtvsotv, M. J, (1936) Distribution of Huid and elxtrolytes and concentration of actornyocin and other proteins in the myocardium of dogs with chronic congestive heart failure. Am. J. Physlol. 187, 483. Con.ovE, E., TRANTHAM, H. V. and Howsutv, R. L. (1938) An instrument and method for automatic, rapid, accurate, and semaitive titration of chloride in biologic samples. J. Lab. clip . Med. Sl, 461 . CaAwt.Er, G. J., GRAY, L,1.®twNa, W. A. and BLANCHARD, J. W. (1966) Blood binding, distribution and excretion of staphylococcal enterotoxin in monkeys. J. Inject . DIs. 116, 48 . Et.wEt.t., M. R., Ltu, C. T., SeERTZBr., R. O. and BEIaEL, W. R. (1975) Mxhanisms of oral staphylococcal enterotoxin B-induced emeais in the monkey . Pr+x . Soc. exp. lllol. Med.148, 424. FtNEaot.n, M. J. (1%7) Interstitial pulmonary edema. An electron microscopic study of the pathology of staphylococcal rnterotoxerrtis in rhesus monkeys. Lab. Invest . 16, 912. Ftatce, C. H. and SvaaAROw, Y. (1923) The colorimetric determination of phosphorus . J. blol . Chem . 66, 373. GaEENr~i,n, L. J. (1974) Surfactant in surgery. Surg, Clip . N. Am . S4, 979. KrRitY, R. R., PERaY, J. C., CAt~ERwoou, H. W., Rutz, B. C. and L®EttMAN, D. S. (1975) Cardiorespirntory efforts of high positive end~tpiratory pt~attrc. Anesthesiology 43, 533. Ltu, C. T. and DELAUTSR, R. D. (1977) Pulmonary functions in conscious and anesthetized rhesus macaques . Ant. J. vet. Res. 38, 1843 . Ltu, C. T. and DUrAULT, D. R. (1977) Effects of intestinal infusion of staphylococcal enterotoxin B (SEB) on water and electrolyte fluxes : possible mechanisms of diarrhea. Physiologist 20, 57 . Ltu, C. T., DELAV7Ett, R. D. and GRtrrttv, M. J. (19760) Pulmonary dysfunction in rhesus monkeys during staphylocoaal B rnterotoxemia. Physiologist 19, 273. Ltu, C. T., GRtrrttv, M. J. and FAtILT