SHORT ARTICLES

STABILITY OF ESCHERICHZA COLZ ENDOTOXIN IN THE RAT GASTRO-INTESTINAL TRACT

I. BERCZI* Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest

PREVIOUS studies in this laboratory (Berczi, Baintner and Antal, 1966; Berczi et al., 1968) have shown that large oral doses of Escherichia coli 089 endoxtoxin are not toxic to rats, and do not induce any tolerance to endotoxin injected intravenously. Nor were animals previously treated with compound 48/80 or X-rays to damage their intestinal mucosa, and subsequently sensitised to endotoxin by lead acetate, killed by large oral doses of endotoxin (Berczi et al., 1968). Clearly one possible explanation of this failure is detoxication of endotoxin by enzymic or some other mechanism. I therefore tried to demonstrate absorption of endotoxin by a more sensitive method-the haemagglutination inhibition (HI) test.

MA- AND METHODS

Experimental animals. Female albino rats, 100f5 g. and 150&10 g. in weight, and 10-day-old chick embryos were used.

Experiment. Twenty rats 150 g. in weight were anaesthetised with ether, and the anus of each was closed with a pursestring suture. Subsequently, 50 mg. E. coli 089 endotoxin was administered to each through a gastric wound. Five rats were killed immediately and 5 each after 6, 12 and 24 hr (group 11). Five normal rats (group I) were used as controls. The intestinal contents of each group of 5 were pooled and processed together. Livers and spleens from the 24-hr group were processed individually. Organs from two groups of rats were each extracted by the phenol-water method 1 hr after the intravenous administration of 10 mg. (group HI) and 100 pg. endotoxin (group IV).

Immune serum was prepared in rabbits with E. coli 089 lipopolysaccharide- casein complex, as described in a previous paper (Berczi, 1967).

Endotoxin was extracted from E. coli 089 by the warm phenol-water method of Westphal and Jann (1965).

Lead acetate, Pb(C2H302)~. 3H20 (Fisher Scientific Company, USA), was injected intravenously in a dose of 5 mg. in 1.0 ml.

Processing of gastro-intestinal contents. The entire contents of the gastro- intestinal tracts of 5 rats belonging to one and the same group were pooled, made up to 500 ml. with saline, mixed with a magnetic stirrer for 1 hr and centrifuged at 3000 r.p.m. for 30 min.; 50 ml. of the supernatant was withdrawn and treated with an equal volume of 90 per cent. phenol at 65"-68"C (Westphal and Jann). The product was dialysed and used for toxicity and serological assays.

Processing of liver and spleen. From the livers and spleens of the rats, 25 per cent. organ homogenates were prepared individually (Omni-Mixer, Ivan-Sarvall Inc., USA) and processed further with 90 per cent. phenol as described by Ravin et al. (1960).

Present address: University of Montreal, Institute of Experimental Medicine and

I. p*i:i. BACT.-VOL. 96 (1%8) 487 2H2 Surgery, Case Postale 6128, Montreal 3, Quebec. Canada.

488 I. BERCZl

Undiluted . . 1 in 10 . . .

1 in loo . .

1 in lo00 . .

Himagglutination-inhibition (HI). Chicken erythrocytes washed three times with saline were sensitised with 100 pg. per ml. of endotoxin at 37°C for 2 hr, washed three times with saline and made up to a 1 per cent. suspension in Verona1 buffer. HI tests were carried out by TaUtsy’s microtitrator technique (Takhtsy, 1955-56). The haemagglutinin titre of the rabbit serum was 1 in 512; it was therefore used at dilutions of 1 in 200 and 1 in 250 in experiments I and 11, respectively. The HI titre of test materials was expressed as the highest dilution still inhibiting the haemagglutinating effect of the serum dilution used.

Toxicity assays were carried out in female albino rats, 100 g. in weight, sensitised with lead acetate (Selye, Tuchweber and Bertbk, 1966) and in 10-day-old chick embryos by the intravenous technique (Berczi, 1967).

contents Ohr 6 h r

515 515 515

015 515 515

015 515 115

015 215 115

--

--

--

--

TABLE I

In-vivo stability of Ercherichia coIi endoroxin in the gastro-intestinal tract of the rat, as titrated in rats sensitised with lead acetate and in chick embryos

Number of animals dyinglnumber of animals tested, in

rats given

I Dilution of samples I intestinal contents from I 1 ratsgivenendotoxin by mouth and sampled at

chick embryos given I

intestinal contents from rats given endotoxin

by mouth and sampled at

RESUL’IS Toxicity tests on intestinal content by both methods at vm’ous times after

administration of endotoxin showed that there was no loss of endotoxin over 24 hr (table I).

The preparations were also assayed for endotoxin by the indirect HI test. Table I1 shows that endotoxin apparently maintained not only its toxicity, but also its serological activity over 24 hr in the gastro-intestinal tract of rats.

In both series, organ homogenates were assayed for endotoxin also by the indirect HI test. Table III shows that organ homogenates obtained from rats treated with endotoxin intravenously displayed serological activity. In organ preparations from rats given 500 times the easily demonstrable intravenous dose (100 pg. per animal), i.e., 50 rng. orally, no endotoxin was demonstrable by the indirect HI test. Liver homogenates from each of groups 1,III and IV were pooled and assayed for toxicity in chick embryo. Table IV shows that such organ extracts are no more toxic to chick embryos than comparable extracts of normal rat liver.

STABILITY OF ENDOTOXIN IN RAT INTESTINE 489

50 mg. endotoxin orally, killed 24 hr later

TABLE II In-vivo stability of serological properties of E. coli endotoxin in gastro-intestinal tract

of rats as assessed by the indirect haemagglutination test

6 7 8 9

Group

- I

- 11

- 111

IV

10 rng. endotoxin intravenously, killed 1 hr later

Haemagglutination-inhibition titre of intestinal content of

11 12 13 14

Experiment 1 1 rats given endotoxin by mouth and sampled at control

rats

I 4 1024

TABLE III Endotoxin assay of organ extracts by the indirect haemagglutination test

Titre of extract of l l I I

Treatment of animals I serial I spleen in liver in

2

I i

L l

I L L

I

f l - -r - I - T-

I-

32 64 128 256

32 I :;: 1 I 1024 64 128

The HI titre of a solution containing 1OOpg. E. coli 089 endotoxin pa ml. was 1 in 128

- = No haemagglutination-inhibition undiluted. in the first series, 1 in 256 in the second.

490 I. BERCZI

Dilution of liver extracts

TABLE IV Toxicity assay in 10-day-old chick embryos of pooled liver extracts from normal rats

(group Z) and from rats given endotoxin intravenously (groups ZZZ and ZV)

rats given 10 mg. endotoxin i.v. 1 hr

(group 111)

normal rats (group I) before killing

Number of chick embryos deadlnumber tested with pooled liver extracts from

Undiluted 1 in 10

1 in lo00 1 in 10,OOO

1 in 100

315 415 115 - -

rats given 100 g endotoxin i.v. r"h;

before killing (group IV

Totals 8/15 12/15 11/15

515 415 3 js 015 015

415 415 3 j 015

DISCUSSION The data from the present experiments show that endotoxin administered by

mouth maintains both its toxic and its serological properties in the gastro-intestinal tract. Closure of the anus at the beginning of the experiment was necessary to prevent elimination of orally administered endotoxin from the intestinal tract, which otherwise would have taken place within 12 hr. Our failure to demonstrate any lethal effect of endotoxin given by mouth was therefore not due to its destruction by intestinal enzymes, but to non-absorption from the alimentary canal.

Examination of organ homogenates shows the same thing. In no case were positive serological results obtained 24 hr after the oral administration of 50 mg. endotoxin. Liver homogenates from rats given 100 pg. and 10 mg. endotoxin intravenously exhibited moderate and high haemagglutination-inhibiting activity respectively. The sensitivity of the HI system used was approx. 0.4 pg. endotoxin per rnl.; on this basis, the endotoxin content of liver homogenates was approx. 200 pg. per ml. in group 111, and approx. 1.6 pg. per ml. in group IV. Although intravenously administered endotoxin was demonstrable in the liver by the HI test, toxicity tests in the chick embryo gave no useful information. It was repeatedly observed that phenol-water extracts from organs not containing endotoxin are themselves toxic for the chick embryo. In the present experiments, liver homo- genates from rats treated intravenously with endotoxin were hardly more toxic than homogenates of normal rat liver. Others (Ravin, Rutenburg and Fine, 1958) have also observed that parenterally administered endotoxin retains its serological activity while it loses most of its toxicity.

SUMMARY Orally administered Escherichia coli 089 endotoxin has been shown to maintain

both toxicity and serological activity in the gastro-intestinal tract of the rat. Therefore failure to demonstrate oral toxicity of this endotoxin is not due to enzymic destruction of endotoxin, but to non-absorption from the intestine.

Parenterally administered endotoxin was demonstrable in phenol-water extracts from spleen and liver of rats by the indirect haemagglutination-inhibition test.

CONCHOIDAL BODIES 491

The toxicity of these extracts for the chick embryo was, however, far lower than their high serological titres would have suggested; indeed it was hardly higher than that of similar extracts from livers of normal rats.

Organ extracts from rats treated with large oral doses of endotoxin gave neg- ative results in the haemagglutination inhibition test.

REFERENCES BERCZI, I. . . . . . . . . 1967. 2. ImmunForsch. exp. Ther., 132,

BERCZI, I., BAI"ER, K., JR, AND 1966.

BERCZI, I., BERT~K, L., BAINTNER, 1968. This Jourd, %, 481.

RAVIN, H. A., ROWLEY, D., JENKINS, 1960.

RAW, H. A., RUTENBURG, S. H., 1958.

SELYE, H., TUCHWEBER, B., AND 1966. J. Bact., 91, 884.

TAK~TSY, G. . . . . . . . 1955-56. Acra microbiol. hung., 3, 191. WESTPHAL, O., AND JAW, K. . . 1965. Meth. Carbohydr. Chemy, 5,83.

303. Zbl. VerMed, ser. B., 13, 570.

ANTAL T.

K., JR, AND VERESS, B. J. Exp. Med., 112,783.

Proc. SOC. Exp. Biol. Med., 97, c . , A N D FINE, J.

AND FINE, J. 436.

B E R ~ K , L.

THE PROPERTIES AND DEVELOPMENT OF CONCHOIDAL BODIES IN SARCOID AND SARCOID-LIKE GRANULOMAS

W. JONES WILLIAMS AND D. WILLIAMS Pathology Department, Welsh National School of Medicine, Royal Infirmary, Cardiff

WE believe that a study of the formation of intracytoplasmic inclusion bodies in sarcoid and sarcoid-like granulomas may be valuable in the study of the aetiology of sarcoidosis. The present investigation is concerned with conchoidal bodies, which together with birefringent crystals form the Schaumann body (Jones Williams, 1960) (fig. 1). These are frequently found in the granulomas of sarcoidosis and chronic beryllium disease, and less often in Crohn's syndrome and tuberculosis (Jones Williams, 1960, 1964). Conchoidal bodies alone are also very occasionally found in lungs without epithelioid cell granulomas, e.g., in coalworkers' pneumo- coniosis (Jones Williams, 1958).

Conchoidal bodies may develop secondary to and around the crystals (Jones Williams, 1960). They have also been reported as occurring around altered tubercle bacilli (Okudaira, Schwarz and Adriano, 1961 ; Vortel, 1962; Dumont and Sheldon, 1965), fungi (Okudaira et al.) and even developing from granuloma- produced protein complexed with mucopolysaccharides (Frizzera and Mancini, 1966). Our present investigations suggest that conchoidal bodies develop from residual bodies, which are end-products of activated lysosomes.

We have previously reported on the properties and nature of residual bodies in sarcoid and sarcoid-like granulomas (Jones Williams and Williams, 1967). The