Physio-pathology of induced endotoxaemia in bovine and its treatment regimen

doi:10.4236/jbise.2013.611135

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J. Biomedical Science and Engineering, 2013, 6, 1077-1084 JBiSE

http://dx.doi.org/10.4236/jbise.2013.611135 Published Online November 2013 (http://www.scirp.org/journal/jbise/)

Physio-pathology of induced endotoxaemia in bovine and

its treatment regimen

Irtiza Nabi1, Digvijay Singh1, Naresh Kumar Sood2

1Department of Veterinary Physiology and Biochemistry, College of Veterinary Science, Guru Angad Dev Veterinary and Animal

Sciences University, Ludhiana, India

2Department of Veterinary Pathology, College of Veterinary Science, Guru An gad Dev Veteri nary and Animal Sciences University,

Ludhiana, India

Email: [email protected]

Received 30 September 2013; revised 2 November 2013; accepted 18 November 2013

permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Endotoxic shock was induced in five apparently healthy

male buffalo calves by i.v infusion of Escherichia coli

endotoxin at 5microgram/kilogram (μg/Kg) body

weight/hour (BW/hr) for 3 hours. Endotoxin infusion

caused clinical signs of restlessness, respiratory dis-

tress, snoring, diarrhoea, profuse salivation along with

the significant hypoproteinemia, hypoalbuminemia and

hypokalemia in all the animals. The animals were ob-

served up to day 4 or death, whichever was earlier.

The treatment with one time intravenous infusion of

hypertonic saline solution @ 4milliliter/Kilogram body

weight (ml/Kg·BW), dextran-40 @ 10 ml/Kg·BW, flu-

nixin meglumine @ 1.1 milligram/Kg·BW (mg/Kg·BW)

and blood @ 20 ml/Kg·BW to these animals alleviated

the clinical signs and significantly raised the circulat-

ing glucose level at 4.5 and 5.5 hrs. The treatment led

to survival of three of the five endotoxemic buffalo cal-

ves. The significant hypoproteinemia, hypoalbumine-

mia, hypokalemia and hypoglobulinemia continued

even after treatment. Gross and histopathologic find-

ings of congestion, haemorrhage, necrosis in vital or-

gans viz., lungs, liver, kidneys, brain and intestines

were suggestive of endotoxin induced hypoxia and

multi-organ failure. Additionally, emphysema and fi-

brinous thrombi in microvasculature of lungs were

salient histopathological findings indicating terminal

respiratory failure in the remaining two dead endo-

toxemic buffalo calves. From clinical signs, plasma

chemistry and pathological lesions, it was concluded

that endotoxemia led to a disruption of critical life

processes, but a timely and effective treatment could

counter these deleterious effects and save precious

lives.

Keywords: Blood; Buffalo Calves; Dextran-40;

Endotoxemia; Flunixin Meglumine; Hypertonic Saline;

Physiology; Pathology

1. INTRODUCTION

Endotoxic shock is an acute circulatory failure occurring

in the presence of severe infection and represents an im-

balance between the body’s oxygen demand and supply.

It is principally of the distributive type, although cardio-

genic and hypovolemic components may also be invol ved

[1]. Endotoxin is an integral component of the outer me m-

brane of gram-negative bacteria and the effects of expo-

sure of host cells to it include the un controlled release of

cytokines and eicosanoids, kinins and other short, me-

dium and long-term reactants that upset the balance be-

tween pro-inflammatory and anti-inflammatory pathways,

causing hypotension, disseminated intravascular coagula-

tion, abortion and death [2]. Endotoxemia is a life-thr ea te n-

ing inflammatory condition, which can lead to shock, mul-

tiple organ failure, suppression of immune system and in-

terferes with wound-healing processes [3].

Despite many limitations, animal models remain es-

sential for the development of new therapeutic regimens

of endotoxic shock, which can’t be replicated by in-vitro

studies [4].

Therefore, the present investigation was planned with

the objectives to study the major physio-pathological

changes during induced endotoxemia in buffalo calves

and the effects of treatment regimen of hypertonic saline

solution, Dextran-40, Flunixin meglumine and whole blo od

infusion on these profiles. The studies could also help in

finding suitable therapeutic regimens applicable in other

species of animals and man.

2. MATERIAL AND METHODS

Five apparently healthy 6-month to one-year-old male

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I. Nabi et al. / J. Biomedical Science and Engineering 6 (2013) 1077-1084

1078

buffalo calves with body weight range of 70 - 140 Kg

were used in the present investigation. These calves were

kept under the good managemental conditions as are

practiced at the university dairy farm. All the animals

were de-wormed and vaccinated against haemorrhaegic

septicemia well before the start of the experiment.

The Endotoxin1 was reconstituted by dissolving it in

normal saline solution (0.9%NaCl) to make a stock solu-

tion of 1 mg/ml. Endotoxin concentration of 5 µg/ml was

prepared by dissolving 1 ml of stock solution in 199 ml

of normal saline to make a total volume 200 ml. The en-

dotoxin was then infused intravenously through jugular

vein @ 5 µg/kg BW/hr for 3 hours, followed immedi-

ately by a rapid infusion of hypertonic saline solution @

4 milliliter/Kilogram body weight (ml/Kg·BW), dextran-

40 @ 10 ml/Kg·BW, flunixin meglumine @ 1.1 milli-

gram/Kg·BW (mg/Kg·BW) and blood @ 20 ml/Kg·BW

Body as one time treatment. The HSS had been prepared

by dissolving 72 gm of sodium chloride in one liter of

double distilled water (7.2% Nacl aqueous solution) and

autoclaved at a pressure of 15 lbs for 20 minutes, 16 - 18

hrs prior to infusion.

The animals were casted in right lateral recumbency

on operation table. Before endotoxin infusion, an area

over jugular furrow was shaved and disinfected. The lo ca l

anesthetic Lignocaine (2%) @ 90 - 120 ml was injected

subcutaneously and intra-muscularly before catheteriza-

tion of the jugular vein and the carotid artery. The blood

samples from jugular vein of the experimental buffalo

calves were collected in heparin immediately before and

after 1.5, 2.5, 3.5, 4.5, 5.5, 6.5 hrs of the start of endo-

toxin infusion, follow ed by samples at 24 hr of last sam-

ple of day 1, upto 4th day. The following physiological

constituents like the plasma total protein, acute phase

proteins like albumin, fibrinogen, globulins, alkaline phos-

phatase, creatinine, glucose, sodium, potassium, calcium

and phosphorus were estimated by dry stat clinical chem-

istry analyser2. Plasma fibrinogen was estimated using a

portable refractometer by comparing the protein in non-

heated plasma and that in the respective sample heated to

56˚ to 58˚ Celsius for 3 minutes [5]. The data so gener-

ated was pooled and analysed with CRD Anova [6]. All

the values obtained were compared with the normal pre-

infusion values within the group. The study design and

animal experimental protocols were approved by the In-

stitutional Animal Ethics Committee (IAEC) of the Uni-

versity.

3. RESULTS AND DISCUSSION

3.1. Clinical Signs

Clinical signs observed due to endotoxemia consisted of

restlessness, respiratory distress, forceful abdominal res-

piration, diarrhoea and profuse salivation. Three endoto xe-

mic buffalo calves out of five animals survived beyond

the observation period, whereas, the remaining two buf-

falo calves d ied duri ng the observ ation pe r iod.

3.2. Clinical Chemistry

The results of the physiological p rofiles estimated during

present investigation are presented in Table 1 and Fig-

ures 1-3.

The normal mean pre-infusion total protein was found

to be 6.62 ± 0.17 g/dl, which is close to 6.82 ± 0.24 g/dl

reported by Sobti et al. (1981) [7], 6.40 ± 0.19 g/dl [8],

but lower than 7.54 ± 0.25 g/dl [9]. In the present study,

however, a significant (P < 0.05) hypoproteinemia was

observed at 3.5 hr after the endotoxin infusion (Figure 1).

[10,11] also observed hypoproteinemia on E. coli endotoxin

infusion in bovine calv e s.

The hypoproteinemia as observed in present investiga-

tion was perhaps due to increased protein catabolism

coupled with decreased ability of anoxic liver to metabo-

lize amino acid to synthesize protein, besides sequestra-

tion of plasma proteins into tissues [8].

A significant (P < 0.05) hypoproteinemia continued

even after treatment with HSS, Dextran-40, flunixin me-

glumine and blood at 4.5, 5.5 and 6.5 hrs., which may be

attributed to the rapid plasma volume expansion and the

redirected splanchnic perfusion following HSS infusion

[12]. In fact, the HSS acutely increases the plasma osmo-

larity and draws intra cellular and interstitial water in to t he

vascular space. There was plasma volume expansion of

3ml for every 1ml of hypertonic saline infused [13].

The pre-infusion plasma albumin was found to be 2.78

± 0.10 g/dl, which is similar to 2.70 ± 0.12g/dl [14] but

lower than 3.29 ± 0.13 g/dl and 3.20 ± 0.19 g/dl [15,16].

A significant (P < 0.05) hypo-albumine mia was observed

throughout th e endotox in infusion (Figure 1). Hypoalb u-

minemia persisted even after treatment at 4.5, 5.5 and 6.5

hrs. in a previous study [8]. A significant (P < 0.05) hy-

poalbuminemia in endotoxin infused buffalo calves was

also observed [8]. The fall in albumin can be attributed to

loss of blood and plasma in tissues, besides diarrhoea,

the common manifestations in endotoxic shock. The hy-

po-albuminemia, in turn, might contribute to hypoprote-

inemia [15].

The pre-infusion mean plasma fibrinogen recorded was

0.24 ± 0.04 g/dl, which is lower than 0.30 to 0.8 gm/dl [5]

and 0.35 to 0.60 g/dl [16]. In the pr esent study, only non-

significant post-infusion alterations in fibrinogen level

were observ ed throughout th e experiment (Figure 1).

The normal or pre-infusion mean plasm a globulins were

found to be 3.54 ± 0.22 g/dl, which is close to 3.90 ±

0.39 g/dl [14], but higher than 3.24 ± 0.24 g/dl [15], 2.34

± 0.25 g/dl [17] and 2.79 ± 0.13 g/dl [18]. In the present

1Escherichia coli endotoxin Lyophilized (Phenol extracted) 0111:B4

lipopolysacharide, SIGMA Chemicals USA.

2Dr

stat clinical dr

chemist

anal

ser, Johnson & Johnson, USA.

I. Nabi et al. / J. Biomedical Science and Engineering 6 (2013) 1077-1084

1079

Table 1. Clinical chemistry (Mean + S.E.) at different stages of endotoxic shock and after treatment with HSS, Dextran-40, Flunixin

meglumine and Blood.

Group Endotoxic shock After treatment

0 h 1.5 h 2.5 h 3.5 h 4.5 h 5.5 h 6.5 h Day2

Total protein(g/dl) 6.62 ± 0.17 6.04 ± 0.39 5.96 ± 0.235.20* ± 0.214.44* ± 0.284.50* ± 0.16 4.88* ± 0.07 6.32 ± 0.39

Albumin(g/dl ) 2.78 ± 0.10 2.20* ± 0.08 2.26* ± 0.071.98* ± 0.581.60* ± 0.09*1.58* ± 0.07 1.78* ± 0.11 2.54 ± 0.28

Fibrinogen(g/dl) 0.24 ± 0.04 0.24 ± 0.04 0.44 ± 0.190.40 ± 0.09 0.36 ± 0.04 0.57 ± 0.27 0. 60 ± 0.22 0.64 ± 0.20

Globulins(g/dl) 3.54 ± 0.22 3.60 ± 0.31 3.26 ± 0.402.82 ± 0.27 2.48* ± 0.272.36* ± 0.33 2.50* ± 0.29 3.14 ± 0.39

Glucose(mg/dl) 92.00 ± 2.17 101.60 ± 10.64 93.80 ± 8.26130.20 ± 10.18175.00* ± 12.17154.40* ± 15.20 110.60 ± 17.33 130.20 ± 33.44

Creatinine(mg/dl) 1.52 ± 0.15 1.60 ± 0.14 1.74 ± 0 .181.54 ± 0.09 1.52 ± 0.10 1.56 ± 0.10 1.7 4 ± 0 .23 2.12* ± 0.29

Sodium(mmol/l) 133.60 ± 2.21 131.60 ± 2 .64 134.40 ± 1.72135.40 ± 1.29131.20 ± 2.91130.00 ± 3.74 136.00 ± 2.12 132.60 ± 2.04

Potassium(mmol/l) 3.36 ± 0.15 2.72* ± 0.12 2.78* ± 0.222.64* ± 0.122.40* ± 0.152.38* ± 0.07 2.46* ± 0.11 2.92 ± 0.22

Calcium(mg/dl) 9.82 ± 0.72 8.02* ± 0.34 7.46* ± 0.236.86* ± 0.146.56* ± 0.226.74* ± 0.28 7.28* ± 0.38 7.88* ± 0.43

Phosphorus(mg/dl) 5.16 ± 0.31 5.14 ± 0.16 5.02 ± 0.244.92 ± 0.15 4.58 ± 0.29 4.66 ± 0. 22 4.68 ± 0.30 5.74 ± 0.60

Alkaline

Phosphatase(U/l) 63.40 ± 18.62 70.60 ± 21 .95 71.40 ± 19.3578.00 ± 26.9078.00 ± 15.9074.20 ± 20.95 84.80 ± 21.0 77.80 ± 19.20

No. of animals in group = 5; *Significant at 5% level.

creatinine

0.5

1.5

2.5

0 hr

1.5 hr

2.5 hr

3.5 hr

4.5 hr

5.5 hr

6.5 hr

2 day

time

conc. mg/dl

creatinine

Figure 1. Total protein, Albumin, globulin and fibrinogen dur-

ing and after endotoxic shock and treatment. Figure 2. Creatinine during and after endotoxic shock and

treatment.

study, buffalo calves did not show any significant change

in plasma globulins during endotoxin infusion. After tr eat-

ment, a significant (P < 0.05) fall in globulins at 4.5, 5.5

and 6.5 hrs. was observed, which may be due to the in-

creased protein breakdown as also reflected by hypopro-

teinemia, in the present study. The pre-infusion mean

plasma creatinine observed was within the physiological

range i.e., 1.52 ± 0.15 mg/dl [15], which is also close to

1.10 to 1.30 ± 0.10 mg/dl [12]. A non-significant varia-

tion in plasma creatinine (Figure 2) was ob served during

the intravenous infusion of the endotoxin. However, plasma

creatinine increased significantly after treatment on day 2

of observation in comparison to pre-infusion level. It ma y

probably be due to the protracted endotoxin induced re-

nal damage as observed grossly and histopathologically

in the present study.

glucose

100

120

140

160

180

200

0 hr1.5

hr 2.5

hr 3.5

hr 4.5

hr 5.5

hr 6.5

hr 2

day

time

conc. m g/dl

glucose

Figure 3. Plasma glucose during and after endotoxic shock and

treatment.

The normal mean b lood gluco se observed w as 92.00 ±

2.17 mg/dl, which is higher th an 67.50 ± 1.74 mg/dl and

75.30 ± 0.97 mg/dl. [14], [8]. In the present study, the

plasma glucose level showed a non-significant alteration

during i.v. infusion of endotoxin. However, after treat-

ment, a significant (P < 0.05) hyperglycemia at 4.5 and

5.5 hrs. was observed (Figure 3), suggesting probable

beneficial effects of Dextran-40, which gets converted

into glucose through metabolism in liver. Hyperglycemia

may also be due to release of epinephrine due to the

stress of ca theterization, besides sh o ck.

The pre-infusion mean plasma calcium observed was

9.82 ± 0.72 mg/dl, which is within the physiological

range of 8.7 - 11.4 mg/dl [15] and 9.7 - 12.4 mg/dl [19].

In the present study, a significant (P < 0.05) hypocalca-

emia was observed throughout the experiment as also

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I. Nabi et al. / J. Biomedical Science and Engineering 6 (2013) 1077-1084

1080

reported previously [20]. The pre-infusion mean plasma

phosphorus observed was 5.16 ± 0.31 mg/dl., which is

close to physiological value of 5.6 - 6.5 mg/dl [19] and

the plasma phosphorus did not show any significant va r ia -

tion during and after the intravenous infusion of endo-

toxin and the treatment.

The pre-infusion mean plasma sodium observed was

133.6 ± 2.21 mmol/l, which is close to 136.6 ± 5.51

mmol/l [16 ], 134.48 ± 4.07 mmol/l [9] an d 133.40 ± 3.19

mmol/l [20]. However, a generalized non-significant in-

crease in sodium was noticed throughout the period of

experiment as also recorded in endotoxemic cows [21]

and buffalo calves [14], previously. In the present inves-

tigation, the mean sodium level at the end of the obser-

vation period was almost equal to pre-infusion level. Ab-

sence of any significant increase in plasma sodium even

after infusion of HSS seems to be advantageous as it

makes the infusion of HSS safe [22].

The pre-infusion mean plasma potassium ranged be-

tween 3.36 ± 0.15 mmol/l, which is close to 3.60 ± 0.2

mmol/l [21] but higher than 2.28 ± 0.18 mmol/l [9]. A

significant (P < 0.05) hypokalemia was observed at 1.5,

2.5 and 3.5 hrs. i.e., throughout the endotoxin infusion

and even after treatment at 4.5, 5.5 and 6.5 hrs. This fall

in potassium level may be regarded as an attempt by the

body to sequester potassium as a part of the mechanism,

whereby endotoxins promote the release of endogenous

pyrogens from leucocytes. Decrease in potassium level

could also be attributed to release of histamine during en-

dotoxic shock, which increases the capillary permeability,

besides regulating the secretion of adrenaline and nor-

adrenaline together [23]. Hypokalemia after treatment

can rather be attributed to rapid volume expansion fol-

lowing HSS infusion [12]. Decrease in plasma levels of

sodium and potassium may furthermore be attributed to

the greatly diminished active transport of sodium and po-

tassium across the cell membrane [23].

The pre-infusion mean plasma alkaline phosphatase

was 63.40 ± 18.62 U/l, which is lower than 173 ± 40 U/l

as reported by [12]. A non-significant variation in plasma

alkaline phosphatase was recorded throughout the period

of observation.

4. PATHOLOGICAL LESIONS

Two out of five endotoxemic buffalo calves that died

during observ ation period were su bjected to necrop sy ex-

amination in order to study the gross and histopathologi-

cal changes.

4.1. Gross Lesions

Gross lesions varied from mild to clear cut haemorrhages

along with congestion and emphysema in lungs (Figure

4) in both the endotoxemic buffalo calves. In addition,

congestion, odema and dilation of intestines (Figure 5),

haemorrhages on its mucosal surface as well as in medi-

astinal lymph nodes and in the gall bladder mucosa were

noticed. Severe congestion leading to reddish discolora-

tion of cortex of the kidneys (Figure 6) was conspicuous.

The gross lesions were suggestive of toxemia, enteropa-

thy and pulmonary insult accompanying endotoxic shock.

Figure 4. Lung-haemorrhages and emphysema.

Figure 5. Intestines-congestion, edema and dilatation with di-

arrheic contents.

Figure 6. Kidney-marked congestion in cortex.

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4.2. Histopathology

Histopatholgically, the lesions were more specific and

the common findings in both the animals were conges-

tion, haemorrhages and emphysema in lungs (Figure 7),

necrotic enteritis (Figure 8) with mononuclear cell infil-

teration, congestion and lower nephron nephrosis in kid-

neys (Figure 9), mild sinusoidal congestion and hepato-

cellular degeneration (Figure 10), besides focal necrosis

in liver. In gall bladder, haemorrhages along with s lough -

ing of mucosa (Figure 11) were also recorded. These

findings indicated muti-organ failure as the cause of death

in endotoxaemia [4]. The small intestine revealed a very

characteristic segmental necrosis of the villi (Figure 8),

possibly related to ischemia or marked hypoxia. Morpho-

logical and functiona l damage in human intestinal epithe-

lium has been correlated to induced iNOS synthesis by

endotoxin causing in crease in epithelial permeability and

secretory diarrhea [24].

Figure 7. L u n g - m a r k e d c o n g e s t i o n a n d e m p h y s e m a ( H . E.X150).

Figure 8. Intestine-necrosis of villi (H. E.X150).

Figure 9. Kidney-diffuse necrosis of tubular epithelium (lower

nephron nephrosis) along with congestion in a glomerulus (H.

E.X300).

Figure 10. Liver-congestion and mild vacuolar degeneration of

hepatocytes (H. E.X300).

Figure 11. Gall bladder-haemorrhages along with sloughing of

mucosa (H.E.X150).

I. Nabi et al. / J. Biomedical Science and Engineering 6 (2013) 1077-1084

1082

Atelactasis, congestion, haemorrhages and emphysema

in lungs, edema in intestinal mucosa along with exten-

sive coagulative necrosis of epithelial cells of villi, dif-

fuse coagulative necrosis of convoluted tubules of kidney

and congestion, haemorrhages with fatty degeneration and

coagulative necrosis in liver was previously observed.

[25] Edema, congestion and haemorrhages in lungs,

nephritic changes in kidney, areas of coagulative necro-

sis of the hepatocytes and necrosis of the intestinal villi

with infiltration of polymorphs was also reported earlier

[26].

In one of the endotoxemic buffalo calves, degenerative

changes of cardiac myocytes along with mononuclear

cell infiltration in perivascular region of the myocardium

(Figure 12) were also noticed, suggesting cardiomyopa-

thy. Hackel et al. (1974) [27] reported myocytic necrosis

and haemorrhage in the sub-endocardial region of myo-

cardium in endotoxaemia.

In the second buffalo calf, congestion, haemorrhages,

neuronal degeneration and mild edema in the cerebrum

(Figure 13) indicated endotoxin induced encephalopathy

[28]. Nagaraja (1979) [10] and Sokkar (2003) [25] also

reported numerous haemorrhages and edema in cerebrum

of endotoxemic calves and rams, respectively. These

changes were suggestive of toxemia related vascular in-

jury, particularly the haemorrhage in the brain might be

of greater relevance.

Fibrinous thrombi in the microvasculature of lungs

were also noticed in one buffalo calf (Figure 14). Naga-

raja et al. (1979) [10] also observed fibrin thrombi in ar-

terioles and capillaries of lungs in endotoxemic calves.

These findings led to the belief that there was setting up

of intravascular coagulation. There are considerable in-

terspecies differences in sensitivity to endotoxin, how-

ever, humans and ruminants show a relatively similar and

Figure 12. Heart-Mild mononuclear infiltration and degenera-

tion (H.E.X300).

Figure 13. Cerebrum-congestion, haemorrhages, neuronal dege-

neration and mild edema (H. E.X150).

Figure 14. Lung-congestion and fibrinous emboli in vessel (H.

E.X75).

enhanced response to endotoxin. Therefore, in the present

investigation, bovine was selected as animal model to si-

mulate human endotoxic shock [4].

4.3. Treatment

Treatment of endotoxemia is difficult because of the nu-

merous mediators involved. There are three possible ap-

proaches in treating endotoxemia. The interaction of en-

dotoxin with target cells can be blocked by inducing tol-

erance, decreasing plasma endotoxin concentrations, or

interfering with endotoxin binding [29]. According to Ei-

cosanoids are responsible for many of the clinical m an if e s-

tations of endotoxemia [30]. NSAIDS inhibit the produc-

tion of eicosanoids and TXA-2. Among NSAIDS, flu-

I. Nabi et al. / J. Biomedical Science and Engineering 6 (2013) 1077-1084 1083

nixin meglumine is the most effective and is anti-endo-

toxic at doses much lower than those required for anal-

gesia. Administration of hypertonic saline leads to reen-

try of extravascular fluid s into the vascular compartment

to produce a more rapid response and marked haemody-

namic effects than conventional use of isotonic solutions

[31]. Endotoxemia also leads to protein loss and reduced

colloidal oncotic pressure. The use of colloid therapy, in-

cluding synthetic and natural colloids, is therefore indi-

cated to mainta in the colloidal o ncotic pr ess ure and to r e-

plenish the protein loss [32]. The fluids are critical in the

pathogenesis and early resuscitation of septic shock. Ad-

ministration of fluids especially colloids and crystalloids

modulate inflammation, improve micro - v asu l ar p erf u si on ,

impact organ function and thereby outcome. The combi-

nation of all these factors might have led to survival of 3

out of 5 calves in the present st udy .

5. CONCLUSION

A perusal of clinical signs, clinical chemistry and patho-

logical lesions in dead calves indicated that there is a

muti-organ damage and pathologies induced by entotoxic

shock are critical for maintenance of life processes. If the

damage becomes irreparable, the animal may die. How-

ever, constituting timely therapeu tic regimens co ns is ti n g

of hypertonic saline solution, dextran-40, flunixin me-

glumine and whole blood infusion as used in the present

study could counter or reverse some of the deleterious

effects of endotoxins and th ereby save lives.

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