44

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2/14 Some Remarks on the s in Gnotobiologieal Experiments

- - {:ontrol Animals

O. V. CHAKHAVA, Laboratory of Gnotobiology, The Gamaleya Institute for Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow The main methodological approach in gnotobiology is comparative experiment in germfree and conventional animals. In some eases one compares germfree and conventional animals per se without additional experimental treatment. Xn other cases both groups of animals are subjected to various experimental treatment. An exception from these two categories of experiments arises when control ammals are also germfree (for instance in studying bacterial interrelationship in vivo in the guts of ex-germfree animals, etc.). We are not going to discuss this group of experiments. As a rule, germfree and conventional controls are fed the same sterilized diet. In this case it is suggested that both groups of animals should have equivalent feeding. B u t such a methodological arrangement comes into conflict with basic gnotobiological findings. To prove this we will mention t h a t first it is axiomatic t h a t the gut mieroflora plays an i m p o r t a n t role in the nutrition and processing of food, some components of which are changed b y the microflora, destroyed or utilized. Secondly, sterile feeding of conventional animals leads to a reduction of species composition of the gut microflora. As a results such animals become somewhat different from the conventional and assume some characteristics of germfree animals, e.g. have an enlarged coecum, decreased weight o f lymph nodes and some morphological characteristics of lymphatic tissue, etc. The t w o reasons mentioned above make it necessary that the compared groups of animals, germfree and conventional, do not receive an equivalent diet. These differences are increased b y the fact that germfree and conventional animals differ from each other in nutritional needs, e.g. in the vitamin B group and ascorbic acid. Then t h e y differ in absorption, the proximal distal gradient of distribution of intestinal enzymes, basic metabolism, blood circulation and the level of oxycorticosteroids in blood. Thus, when comparing groups of animals, germfree and conventional, with no adequate feeding, it must be realized t h a t in fact t h e y have different nutritional conditions, which leads to the formation of a different immunological and physiological state. Therefore it seems t h a t there is a need for special diet for conventional control animals of the corresponding species, and also a need to prevent reduction of the gut microflora b y inoculation of sterilized diet and b y a periodic contact between control conventionals and stock animals. B y these measures it might be easier to differentiate between experimental results deriving from the absence of microflora and those from a changed physiological and nutritional state. 2/15 The Use of Gnotobiotie Rats for Eharaeterization of Digestibility of Maltitol H. GARTNER, H.-J. ZUNFT, J. SCHULZE, W. MULLER, F.-K. GRUTTE, Central In-

stitute of Nutrition of the Academy of Science, Postdam-Rehbr~tcke, GDR A t t e m p t s to decrease the consumption of sucrose led to the search for new sugar surrogates. The sugar alcohol maltitol, a disaccharide consisting of glucitol and

1979

ABSTRACTS

45

glucose, is formed b y catalytic hydration of maltose. Maltitol is distinguished b y a sweetness of 75 % t h a t of sucrose. :Preparations of maltitol have already been in use for several years, especially as the literature of patents excluded the absorption of maltitol (1969, 1973). Dahlquist and Telenius (1965), later Wfirsch (1975) and Rennhard (1976), however, reported t h a t the enzyme hydrolysis of hydrated maltose derivatives was possible b y the maltases of the small intestine. Our investigations were carried out in an attempt to obtain information about the use of maltitol as a sugar surrogate.

Methods and materials The qualitative determination was done b y TLC on silica gel (kieselguhr 4/6 with 2-propanol--e?~hyl a c e t a t e - - w a t e r 83 : 11 : 6). For the quantitative determination maltitol was hydrolyzed b y e-glucosidase with a subsequent enzyme determination of glucose. The mucosal disaceharidases from the small intestine of men, rats and rabbits were examined for their ability to hydrolyze maltitol after solubilization with Trit o n X-100 and subsequent centrifugation. The gnotobiotie experiments were carried out with Wistar rats, strain Wag/rij germfree and monoassociated with aerobic bacilli. The number of germs in the faeces of monoassociated rats are below 105. I n vitro controls did not indicate a measurable consumption of maltitol at bacterial count. 0 . 1 % polyethylene glycol 4 000 served as an unabsorbable marker.

Results 1. Mucosal disaccharidases of the small intestine hydrolyze maltitol with a relative sphtting activity of 1 (rabbit), 5 (man) and 19 (rat). 2. The splitting of maltitol in everted sacs of the small intestine of rabbits occurred slowly in comparison with sucrose. 3. During perfusion the small intestine of monoassociated rats (N = 5) absorbed 20 % of the maltitol applicated. The rate of hydrolysis m a y be compared with that of lactose in perfusion-lactase adapted rats. 4. Germfree rats (N = 5) were given a 30 % solution of maltitol with a gastric tube. The animals were killed 2 h later. Subsequently, the ratio of maltitol polyethylene-glycol was analyzed in the content of the stomach and in the proximal and distal sections of intestine. The ratio decreased from 1.0 in the stomach to 0.42 in the first and 0.31 in the second section of the intestine. 5. Oral intake of 60 g maltitol caused in the two samples an increase of the glucose concentration in the serum up to a m a x i m u m of 20-- 30 rag/100 ml. The starting value has not been reached after 3 h. Doses of 35 g maltitol per d a y applied over a period of 10 d were tolerated b y the patients and utilized b y the organism.

Conclusion All these results indicate the enzyme degradation and the absorption of maltitol. The absorption takes a slower course than with sucrose, b u t it is obviously complete. As maltitol supplies She organism with energy equal to that of sucrose, it is not suitable as a sugar surrogate in low-energy products.

46

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Voh 24

REFERENCES D~_~z~uIsT A., TET.~.NIUS V.: The utilization of a presumable low-cariogenic carbohydrate derivative. Acta Phyaiol. Scand. 63, 156 (1965). REN~HA~D H. H., BI~NCHI~. J.: Metabolism and caloric utilization of orally administered maltitol-14 C in rat, dog and man. J. Agr. Food Chem. 24, 287 (1976). WiJ~scH P.: Food Sci. Technol. Abstr. 7, 88 (1975).

2/16 The Catabolism of Uracil and Cytosine in GermIree Animals F. NOVAK, I. KOZAK, Z. DIENSTBIER, Institute of Biophysics and Nuclear Medicine, Charles University, Prague and M. VITOLS, A. GRIZqBERGS, V. KRI~VINA, L. VITRI~AK, Faculty of Biology, Latvian State University, Riga, USSR The catabolism of uracil and cytosine were st~udied in germfree and conventional animals b y means of total balance of excreted radioactivity after peroral and intraperitoneal application of laC-labelled compounds. There was no significant difference in uracil conversion to carbon dioxide between guinea-pigs, rats, mice and golden hamsters. 14C02 was collected for 24 h which resulted in obtaining 65 ~/o of the dose given initially. At the same time, there was no difference between conventional and germfree animals in the uracil conversion to carbon dioxide. Intermediates of the reductive p a t h w a y of uracil catabolism b u t also a little amount of barbituric acid, the product of an oxidative p a t h w a y were identified. After peroral application of laC-cytosine there is a difference in the conversion to carbon dioxide b e t w e e n species under study. Most radioactivity was excreted b y guinea-pigs (52 ~o), less b y rats (18 % and by mice (12 ~o) of the initial dose. Only 11 ~ of the initial dose was degraded to carbon dioxide in guinea-pigs after intraperitoneal administration of 2-14C-cytosine. After peroral application 19 ~o of the radioactivity was found in guinea-pig urine. After intraperitoreal administration, 75 ~/o of the dose was found in the urine. 14C-Labelled cytosine, uracil and ureidopropionie acid were identified in urine after both modes of application. 1 ~o of 2-14-C-cytosine was converted to carbon dioxide. All radioactivity in urine was identified as unchanged cytosine.

2/17 The Catabolism of Exoflenous Adenine and Adenosine in Conventional and Germfree Mice l:~. ZVILNA, M. VITOLS, Faculty of Biology, Latvian State University, Riga, USSR, and F. NOVAE, Z. DIENSTBIER, Institute of Biophysics and Nuclear Medicine, Charles

University: Prague

The catabolism of adenine and adenosine were studied in germfree and conventional mice (Lobund L a b o r a t o r y Swiss Webster) b y means of balance of excreted radioactivity after peroral application of 14C-labelled compounds. There was a difference in ~he conversion of 8-14C-adenine and UJ4C-adenosine to carbon dioxide between conventional and germfree mice. Germfree animals expired 2.8 ~o radioactivity of initial adenosine dose as carbon dioxide in 24 h. Conventional animals

1979

ABSTI~ACTS

47

converted 23.5 ~o radioactivity of initial dose to carbon dioxide. On administering u_laC-adenosine to germfree mice the expired carbon dioxide contained 12.3 % radioactivity of ~he initially given dose. In conventional mice 46.6 ~ of radioactivity accumulated as carbon dioxide after peroral application of U-~dC-adenosine. Analysis of urine from conventional mice revealed tha~ most urine-excreted radioactivity after peroral application of adenine and adenosine was in the form of allantoin. The difference in conversion of both compounds to carbon dioxide between conventional and germfree animals can be explained by participation of intestinal microflora in the catabolic pathway.

2/18 Serum Proteins in Conventional (CVN) and Germfree (GF) Miniature Minnesota Pi91ets J. TrtXV~EK, L. MAN~)~L, Laboratory of Gnotobiology, Institute of Microbiology,

Czechoslovak Academy of Sciences, 142 20 Prague 4 Serum proteins of miniature Minnesota suckling piglets from a caesarean-section derived herd kept under strict health control were investigated using agar electrophoresis and compared with GF piglets of the same breed. In GF piglets, a significantly lower level of total nitrogen was found. In both groups, the albumin level showed a decrease during the observed period: no remarkable difference was found between the groups after birth, in CVN pigs the albumin level became higher beginning the 21st day. A fundamental difference was observed in the alphabetoprotein which in CVN piglets decreased to zero during first 14 days but remained constant in GF animals. This finding was ascertained also by the radial immunodiffusion method using specific antisera. The level of the alpha1 fraction was higher in GF piglets, whereas the beta fraction was up to the 21st day lower in comparison with CVN piglets. No gamma globulin was established in GF piglets during the whole period by used method. In n~tive, non-concentrated sera of newborn piglets, only a small amount of IgG was detected. During the whole observed period, suckling piglets showed significantly higher levels of IgG, IgM and IgA. The level of IgG in GF miniature Minnesota piglets kept during the 1st month on a constant level; IgM and IgA was no~ detectable until the 2rid month after birth.

2/19 Solid Diets Used for Rearin 9 Germfree Co]ony Rats and Its Effects on Reproduetion 1~. ~T~PX~xovX, Laboratory of Gnotobiology, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4 Various diets have been used for weaning germfree rats. All the used diets were in fact modifications of a practical diet L-356 or a synthetic diet L-283. Using the diet L-356 and L-462, germfree rats have been reared in the Lobund laboratory for many generations without any symptomps of deficiency abnormalities. The practical

SECTION 2

48

Vol. 24

diet L-462 was replaced due to its high cost b y the more advantageous diet L-485 (Kellogg and ~vVostmann 1959). B y rearing germfree rats in our conditions, the modified diet L-485, called 01 (without food methionine) was used. We found t h a t rats reared on this diet were not fertile in the F1 generation. Cyclic changes of the vaginal epithelium Were investigated in females b y vaginal smears. The rats were found in a continuous dioestrus. Histologically, the ovary was unfunctional. Corpora lutea or mature follicles were n o t present up to the age of one year. Another diet used was the 02 which was enriched before steam sterilization with 0.5 % of food methionine only. Germfree rats reared using this diet had a normal sexual cycle and at the age of three months became pregnant, farrowed and reared their offsprings. Germfree rats were reared on this diet in our laboratory for nine years. The diet is prepared in pelleted form. The diet was sterilized in sterilization cylinders where it was placed in layers 10--20 mm thick. The diet was sprayed with 30 ~ of' water before autoclaving. The sterilization regime: 70 kPa, 5 120 kPa, 5 evacuation: 70 kPa, 5 s t e a m pressure: 130 kPa, 30 release of steam a n d air follows. evacuation: s t e a m pressure:

min, rain, rain, min,

30 ~ 118 ~ 85 ~ 124 ~

.By investigating the importance of methionine for rat reproduction, the uterus weight, the weight of the placenta and the early resorption of foetuses were mainly studied. Y a m a et al. (1973) found that methionine deficit caused the resorption of foetuses. For maintaining pregnancy, a supplement of 0:5 % methionine was most suitable. In germfree rats, the need for methionine is higher than in conventional animals, for the available methionine and cysteine are destroyed b y steam sterilization. Methionine is decreased b y 22 % and cysteine b y 75 % b y steam sterilization (Pieniazek et al. 1975). In our rearing unit, the food methionine supplement was found to be important for germfree female rat during pregnancy as well as to the age of 6 weeks. REFERENCES KELLOG(] T. F., WOST~IA.5i'5]"B. S.: Stock diet for colony production of germfree rats a n d mice. Lab. A n i t a . Care 19, 812 (1969). PI~I~IAZEK D., RA~:OWS~.A]VI., SZlLLADZlOWA W., GaABAX~EKZ.: E s t i m a t i o n of available methionine a n d cysteine in proteins of food products b y in rive a n d i n vitro methods. Brit. J . N u t r . 34, 174 (1975). YA~)~ Y., KlSgI K., E~IDO S., I~Ioly~. G.: Effects of diets devoid of essential amino acid on pregnancy in rats m ~ i a ~ i n e d b y o~rarian steroids. N u t r i t i o n 101, 207 (1973).

2/20 The Development of Intestinal Lymphatie Tissues in Germfree and Conventional Rabbits l~. ~T]~PANKOV.~, F. KovA~ff, t J. KRVML, Laboratory of Gnotobiology, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4 Animals reared under germfree conditions are free of all microorganisms including the intestinal flora. During phylogeny a mutual relationship between the host and the microorganisms has developed. The intestinal microflora appears to be one of

1979

ABSTRACTS

-Days

6F

14

30

GF

0 O

62

103

CV

9

0~ 0~

47

CV

.

Fro. 1. Development of intestinal lymphatic follicles in rabbits; left appendix, right sacculus rotundus; 13 F germfree, CV conventional.

natural physiological factors of a healthy conventional animal. Tile intestinal lymphatic tissue has developed as a protective barrier which ensures a positive mutual cooperation between the host and the microorganisms and prevents the damage to ~he host. At birth, rabbit lymphatic tissue is characterized b y an underdeveloped follicular apparatus which consists only of reticular cells and of foci of erythro- and myelopoiesis. From the 4th d after birth, lymphocytes start to appear in the appendix; the first organized lymphatic tissue was found in conventional rabbits on the 10th d a y after birth (Areher et al. 1964). In our experiments, we evaluated histologically the size of lymphatic follicles of conventional and germfree rabbits 14, 30, 62 and 103 d after birth. We observed that in germfree rabbits the lymphatie tissue of the appendix possessed the follieular strueture already after 14 d; no ]ymphatie follicles developed up to 62 d after birth. In conventional rabbits, the lymphatie follicles were found to be 3 - - 5 times as big as follicles of germfree rabbits (Fig. 1). The saeculus rotundus of germfree rabbits had no follicular organization after 14 d and first follicles were detected after 30 d. L y m p h a t i e follicles in the sacculus rotundus of germfree rabbits found after 103 d were comparable to those of conventional rabbits at 30 d. In germfree rabbits, no germinal centers could be demonstrated in t h e appendix and the saeeulus rotundus during the whole observation period. Besides the overall characteristics of the ileum, the saeculus rotundus and appendix, we attempted to establish the number of lymphoeytes migrating through the epithelial layer of the ileum, using the method of Andrew (1965). We determined the localization of lymphoeytes in the ileal epithelimn in the basal, intermediate and apical position. We found t h a t in germfree rabbits most lymphoeytes were in the basal position (14, 30 and 62 d). After 103 d, there was a shift of lymphocytes into the intermediate position. In conventional rabbits, lymphocytes were found after 62 and 103 d in the apical position. In addition, the proportion of lymphocytes in t h e total population of intestinal cells was significantly lower in germfree rabbits as compared to eonventional control animals. The only exception was the initial phase of the postnatal development when in germfree, artificially fed rabbits the proportion of lymphocy~es after 14 d was higher than in conventional, breast-fed rabbits (34 % in germfree, 1 6 . 1 % in conventional rabbits). We presume that the high percentage of lymphoeytes in germfree rabbits is related to the artificial diet used for feeding these animals.

50

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Vol. 24

BEFE BENCES ARCHER O. K., SUTHERIA-ND D. E- R., GOOD 1~. A.: The developmental biology of lymphoid tissue in the rabbit. Consideration of the role of t h y m u s and appendix..Lab. Investig. 13, 259 (1964). A~D~EV W. : Lymphocyte transformation in epithelium. J. 1Ve~t. Cancer Inst. 35, 113 (1965).

2/21 Haematolo9ieal Differences between Germfree and Conventional Miniature Piglets L. MANDEL, J. TRXVNI~EK, Laboratory of Gnotobiology, Institute of Microbiology,

Czechoslovak Academy of Sciences, 142 20 Prague 4 The red and white picture of peripheral blood taken by a heart puncture was investigated in germfree and conventional suckling piglets of a miniature Minnesota breed from birth to eight weeks of age at weekly intervals. Twelve germfree piglets of both sexes descending from four litters were delivered by gnotobiotic hysterectomy and fed by a milk-type diet in sterile isolators as described by Trs et al. (1966). Conventional suckling piglets were kept with their sows in concrete pens. No remarkable differences were found in the erythrocyte count and in haemoglobin which showed a steady increase during the whole observed period in both groups of piglets. However, whereas the haematocrit values increased in conventional piglets from an average of 35 % after birth to 45.6 ~ at the end of the 8th week, in germfree animals these values remained on a newborn level to the end of the observed period. Thus, the mean corpuscular volume of erythrocytes in germfree piglets has kept the newborn size (about 60 ~mS) but increased to 84 ~m 3 in normal conventional animals. Such established normochromic microcytosis in germfree piglets which resembled the post-birth stage of development needs further investigation. No significant differences were established in the thromboc3r counts which steadily increased from about 310 000/mm~ to a maximum of about 400 000/mm 3 in the 5th week and then slightly decreased in both groups. A large difference occurred in the leukceyte count between germfree and conventional piglets: whereas the number of leukocytes in conventional piglets reached about 12 000/mm 3 during the first week after birth and maintained this level during the whole observed period, in germfree piglets the leukocyte count remained on the post-birth starting level (bout 5 000/mmS). The differential count did not show significant differences in the lymphoeyte/granulocyte ratio when both groups were compared. Important differences were found by evaluating the types of neutrophil granuloeytes; as a rule, almost only adult, segmented forms were found in germfree piglets whereas a normal distribution was established in conventional animals. These findings reflected well the decreased continuous antigenic stimuli in germfree pigs when the microflora is absent. In addition, the activity of some haemocoagulation factors was investigated. I t was stated that the synthesis of K-vitamin dependent factors, 'particularly of prothrombin and F VIII, took place in germfree K-vitamin deprived piglets to the 17th day after birth only; 10 mg of vitamin Ks given i.m. weekly kept the blood coagulation activity on a level found as a rule in conventional suckling piglets. REPEBENCE TI~Vl~f(~E~r J., MAND~L L., LA~C A., Rf~I~x.~ I~. : l~earing of germfree piglets. (In Czech) ~s. fyMologi~ 15, 140 (1966).

1979

ABSTRACTS

5|

2/22 Membrane-Bound Enzymes of Lymphoid Tissues. I. ATPase and ~-Glutamyl Transpeptidase Activities of Intact Cells durin 90niogenelie Development of Germfree and Conventional Pigs H. KovAf~, F. KovA~fT, L. MAiD,L, Institute of Microbiology, Czec/~oslova]c Academy of Sciences, 142 20 Prague 4 The interaction of external molecular effectors with plasma membranes of cells generates physiological signals activating metabolic processes, t h a t may result in cell specialization or dedifferentiation and cell proliferation. The membrane-generated signal (e.g., antigen, lectin, hormone) stimulates cellular transport and macromolecular synthesis, eventually culminating in cell division. It is supposed that membrane enzymes, such as Na, K-ATPase (Na +, K+-stimulated, Mg2+-dependent ATP-phosphohydrolase; EC 3.6.1.3) may also participate in membrane regulatory phenomena during mitogen interaction (Coulson et al. 1974, 1977; Barnett et al. 1974; Lelievre et al. 1977), though the most important function of this enzyme is to maintain osmotic equilibrium (Skou 1965). The other membrane-bound enzyme, T-glutamyl transpeptidase (y-GTPase), is found in a great many cells (review: Meister et al. 1976) and recently it has been studied and demonstrated in lymphoid cells (Novogrodsky et al. 1976). This enzyme also plays a physiological role in amino acid transport (Meistcr et al. ]976). Some relationship of the enzyme to the synthesis and secretion of immunoglobulins by lymphoid cells is considered (Novogrodsky et al. 1976; Litwin et al. 1974). Germfree (GF) piglets are characterized by morphological and immunological specificities during their ontogenetic development (Sterzl and Silverstein 1967; Kruml et al. 1969). The aim of this report is to describe the relationship of biochemical changes, represented here by the activity of membrane-bound ATPase and y-GTPase in intact thymus and lymph node cells to morphological changes in these organs during the ontogenetic development of GF minipigs. Materials and Methods

Cell suspensions were prepared from t h y m u s and lymph nodes of GF and conventional (CV) miniature Minnesota pigs at three age intervals -- the 15th, 30th and 6Oth day of postnatal life. Some experiments were performed on 86-day-old GF minipigs fed with a non-antigenic synthetic diet (Abstract 1/10). The cell suspensions were obtained by dissociation of small tissue fragments in isotonic salt medium with 1 % Ficoll and then the cells were washed three times. The activity of enzymes was estimated immediately. The released inorganic phosphate was determined according to Bonting et al. (1961). The activity of Na+K+-ATPase was calculated as the difference between the total ATPase activity and ouabain-resistant Mg~+-ATPase activity. The enzyme activity was expressed in nmol of released inorganic phosphate per hour per 106 cells. The y-GTPase was estimated according to Novogrodsky et al. (1976) wi~h L-y-glutamyl-4-nitroanilide as substrate. Activity units were nmol of product (4-nitroaniline) formed per hour per 106 cells. .Results and Discussion Thymus. The decrease of activity of ATPase and y-GTPase was observed in thymus cells of GF and CV minipigs during the postnatal development. These findings

~2

SECTION 2

u

24

are probably connected with the regressive function of thymus. However, the ATPase activity of 15-day-old and 30-day-old and y-GTPase activity of 15-day-old GF animals was significantly higher in comparison with CV control animals. Lymph nodes. Na, K-ATPase activity of mesenterial lymph nodes of GF minipigs was not changed significantly at the tssted age intervals in comparison with CV controls. Na, K-ATPase activity of peripheral l y m p h nodes of GF animals was lower at the tested age intervals comparing to CV controls. There is evidently a falling tendency of Na, K-ATPase of peripheral lymph nodes during development of both experimental groups, i.e. in GF and CV minipigs. In mesenterial lymph node cells, insignificant diff,erences of y GTPase activity were found between CF and CV animals at tested age-intervals. In peripheral lymph node cells, y-GTPase activity was elevated in both groups during postnatal life. But the increase of y-GTPase activity was markedly slower in GF minipigs. Certain experiments were performed on GF pigs fed with the non-antigenic diet. It was shown that the absence of natural milk antigens affected markedly the activity of ATPase and y-GTPase in t h y m u s and l y m p h node cells from 86-day-old GF minipigs. The values of enzyme activities in both organs were similar to those in 15- or 30-d-old GF minipigs fed with milk diet. The question was whether a relationship exists between the mentioned biochemical data and the morphological and immunochemical findings in other reports. Our experiments indicate some retardation of Na, K-ATPase and 7-GTPase activities of GF lymphoid organs (thymus and lymph nodes) during postnatal development of GF minipigs. Morphological studies (I~eview 2/13) show also retardation of the development of lymphoid population of primary and secondary lymphatic organs. These studies are supported by data of Jaro~kovs et al. (1973) that B lymphocytes in GF minipigs carry only IgM immunoglobulia receptors which are typical of foetal and neonatal periods of life. REFE RENCES B . ~ a ~ ~r P,. E., S ~ r ~ P~. E., F~Ro ~r L. T., K~RsEr J. 1~. : Evidene~ t h a t mitogenic lectins induce changes in l y ~ p ' a ~ y ~ m~mbraae fluidity. Yatur~ 249, 465 (1974). B o ~ S. L., SE~o~ K. A., I-I~vc~:[~s N. H.: Studies on sodium-potassium-activated adenosine triphospharaoh. I. Q l ~ i ~ t i v o distributior~ in sovoral tissues of the cat. Arch. Biochem. Biophys. 95, 416 (1961). '' i ..... CO~rL~o~ A_. S., Z ~ . t r . ~ V. I~., C o ~ 1~., Ga~e~" 1~. B., S~.,~so~ E. B., S~u~w~Y N. E.: Lymphocytem~mbcaav ad~a~inv t r i p h ~ p h a t a s c . Lancet 1017 (1974). Adeao~ia) tripl~a~phata~ lo~tcc[ c a uas~imulatod h u m a n small lymphocyte cell membranes. Quart. J. Exp. Physiol. 62, 181 (1977). J~_ao~ov~ L., Ta~.a~cHkvs~:~ I., ~ i H a I., Kov_~fcf~ F., K o ~ g s M.: Immunoglobulin determinants on lymphvuybo ia g~rmt'roo piglets. Eur. J. I m r a ~ 9 1 . 3 , 818 (1973). K a ~ g 5 J., L~DViK J., T~samg~vsE~2 I., M a ~ . L L., Kov~-f~ff F.: Morphology of germfree piglets. Folia Miorobiol. 14, 441 (1969). L~.h~.~a~ L., P~R~,v A., Cm~aLZ~Xo~z D., S H ~ . e ~ D 5. R.: :Plasma membrane studies on drug sensitive and r~sis~aat o~ll liars. I. Cross resistance and membrane enzyme coordination (Ouabain/cA~[P/ /NaeK:~ATPa~/adenylat~ eyelash). Exp. ~ell Rss. 104, 191 (1977). Lrrwi~ S. D., H i ) ] : r ~ o ~ T. I-L, LI~ P. K., K ~ R D D., C~.V~ H. : ][mmunoglobulin expression of ceils from hum~u lym?hvbia~Loid lines. I L Interrelationship among surface, cellular and secreted immuneglobulins. J . I r a ~ r ~ l . 113, 668 (1974). ~r 9 ~-., T ~ . S. S., P~9~ L. L.: Membrane-bound y-glutamyl transpeptidasc, p. 315 in A. Martonosi (E1.): Th~ Er~,,~r~,~ of B;~9~ojieal MaraSrar~ss, Vol. 3. Plenum Publ. Corp., New York 1976. N)VO~ODSm~" K., T&T~ S. S., NI~.ts~a A.: ~'-Glu~amyl transpoptidaso, a lymphoid cell-surface marker: relationship to bla~togoa~sis, differentiation, and neoplasia. ~Proc. Y a t . Acad. Sol. U S A 73, 2414 (1976). Smov 5. C.: Eazymv,tic basis for active traaspor~ of Na + and K + across membrane. Phyaiol. Rev. 4~, 596 (1965). ~" ~r~.~z5 ~., St~.v~au~.t~ A. M.: D~velopmontal aspects of immunity. Adv. Immunol. 6, 337 (1967).

1979

ABSTRACTS

53

2/23 Functional

Alterations

o f Lymphoid Sys[em i n Germfree Mice

P. AI~DERLIK, I. SZEIr S. B_~NOS, L. BEREK, B. RADNAI, Institute of Microbiology and Department of Orthopaedics, Semmelweis University of Medicine, Budapest and Istvdn Hospital, Budapest Germfree (GF), COBS (cesarean-originated, barrier-sustained) and conventional (CV) CD~-I male mice (Charles River Breeding Laboratories Inc., Wilmington, Mass., USA) were used. D a t a of Table I indicate the u n d e r d e v e l o p m e n t of the lymphoid organs in GF mice. Histologically the t h y m u s - c o r t e x in G F mice was cytopenic with minimum l y m p h o i d cells, t he spleen was also cytopenic and germ/native centers were almost absent. T h y m u s and spleen in CV mice showed normal structure, while the t h y m u s - c o r t e x in COBS mice was r a t h e r cytopenic, the spleen was normal. Cold stress. Up o n exposure of l0 GF and 10 COBS mice aged 28 d at -~4 ~ temp e ra t ur e for 4 h a lymphopenie reaction developed in COBS, b u t not in GF mice. F o u r GF mice died 4 h after stress. B y histology no germ/native centers were found in the spleen of animals succumbing to cold stress, while those possessing germ/native centers survived. LCM virus infection. LCI~ virus (100 LD50) was injected intracerebrally in 20 GF, 20 COBS and 20 CV mice, all aged 42 d. All of t he CV, 85 % of t he COBS, but only 20 % of th e GF mice developed neurological sym pt om s and died 7--13 d after infection. B o th the brain a nd the blood samples of the animals sacrificed on the 21st d contained LCM virus b u t l ym phoc yt i c infiltration of the lcptomeninx b y histology could n o t be demonstrated. Survival of the GF and of the COBS mice as well as histological findings also indicated a deficient cellular-type i m m u n e reaction to LCM virus infection. Investigation of the bones. X - r a y e x a m i n a t i o n of 10 GF and l0 COBS mice revealed shorter t u b u l a r bones, thinner cortices in GF t h a n in COBS mice aged 28 d. Radiomicrometric measurements showed a considerable (42.5 %) r e t a r d a t i o n of t he femoral cortices in GF animals. Histology: t he distal ends of t he femur in the epiT~Lm I. Data of the lymphoid organs (A), lymphopenic reaction (B), lymphocytic choriomeningitisvirus infection (C), and radiomicrometrie measurements of the femur (D) in germfree, COBS, and conventional mice Characteristic A absolute lymphocyte count per nl relative lymphoid organ weights, % of spleen of thymus B absolute lymphocyte count per nl +4 ~

C lymphocytic choriomeningitis, % D sum of femoral cortical thicknesses per nl

Mice germfree

COBS

conventional

1.0 4- 0.4

3.8 4- 0.5

6.0 • 1.0

1.7 4- 0.4 1.7 4- 0.6

4.7 -t- 0.5 1.7 4- 0.5

5.8 • 0.7 2.8 -t- 0.6

0.5 4- O.3 O.6 4- O.3 h 2O

3.6 -4- 0.8 1.2 4- 0.8

0.23•

84 0.394-0.05

I00

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physeal trabeeules of GF mice were slim and thin, with a widening of the intertrabeeular spaces. The growth plates were thinner and the number of cells was lower in each layer. Hyaline pillars were sparse, showing little osteoblastic activity. The results pointed to retarded skeletal growth in GF mice. Conclusion. In mice with an underdeveloped lymphoid system the same alterations of the general and of the immunological adaptation and of osseous growth could be observed as were detected earlier in mice with an impaired lymphoid system. We suppose t h a t the hypofunction or the absence of the function of She lymphoid system m a y be responsible for the disorders.

2/24 influence of Laetobaeilli on the Intestinal Mueosa and Regional Lylnphatie Tissue of Gnotobiotie Rats E. M. GORSKAYA, Laboratory of Gnotobiology, The Gamaleya Institute for Epidemiology and Microbiology, Academy of Medical Sciences, Moscow Germfree Fisher inbred rats were associated with Lactobacillus fermentati and L. plantarum. Morphological and histochemical studies showed lymphocytic, eosinophilic and mast cell infiltrations in the mucosa of the small and the large intestine, a dilatation of the lymph vessels with accumulation of lymphocytes; plasma cell numbers were almost unchanged. Mitotic activity of the cells in the paracortical areas, enhanced migration of the lymphocytes through the endothelium of the postcapillary venules, the formation of lymphocytic muffs around them were found. The plasmocyte reaction had a local character. Antibodies against lactobacilli in the serum were not found or were revealed in low titres by the immunofluorescence technique. After subcutaneous injection of the soluble lactobacillus antigen in the skin of conventional rats positive reactions was noted in 85 ~ , whereas the same skin tests in germfree animals were completely negative. The results indicate the existence of cellular immune response to endogenous lactobacilli in the host.

2/25 Adherence o~ Bacteria to the Intestinal Wall ~ . TALAFANTOVA, ]:. TREBICttAVSKY, V. DLABAC, Laboratory of Gnotobiology, Insti-

tute of Microbiology, Czechoslovalc Academy of Sciences, 142 20 Prague The adherence of Escherichia coli to the intestinal wall of germfree piglets was studied in vitro and in vivo. In vitro methods were used as a model for time histological and electronoptic studies of bacterial adherence and penetration. The everted sac method described b y Wilson and Wiseman uses everted gut segments in Krebs-Ringer hydrogencarbonate solution with pneumoxide and incubation with bacterial suspension. Changes in the morphology of the intestinal tissues were detected in transmission and scanning electron microscopes in intervals fi'om 1 m i n t o 3 h of incubation. The first morphological changes began after 30 rain of incubation without microorganisms. I t thus appears t h a t all experiments with pro-

1979

ABSTRACTS

55

longed incubation times are unphysiological and hence 30-rain exposures were used. E. coli 055 (cell concentration 1/pl) were used for the e v e r t e d sac method. Bacteria were found 10 rain after contamination inside the cytoplasm of epithelial cells. Small gut segments incubated in the same medium were used for quantitative studies of bacterial absorption of three bacterial strains (Smith's P 12 K 8 8 ~ - e n t - - , K 8 8 - - e n t - - and E. coli 055.) Ten minutes of incubation with bacteria was determined as the o p t i m u m period. The segments were 6 times *horoughly washed in saline, homogenized, diluted and the samples were cultivated for 1 d on Endo agar. Maximum absorption was found in newborn piglets with K 8 8 + bacteria. Lesser absorption was observed in segments incubated with E. coli 055 and still lesser absorption was found with bacteria without K88 antigen. Intestinal loops used in experiments in vivo confirmed the difference in the adherence of E. coli strains with and without the K88 antigen. Tissue contaminated with the K88 strain of E. coli showed extensive degeneration of epithelial cells. On the contrary, K88 bacteria were found mainly in the lumen, whereas the fine structure of the tissue was normal Strong adherence of the K 8 8 + strain was observed in the ileum of perorally contaminated piglets. The ileal wall was completely destroyed 2 d after the contamination. The j e j u n u m where lesser adherence was found was sterile at this time. Our results confirm the role of antigens in the mechanism of bacterial adherence and the penetration into the intestinal epithelium.

2/26 Morphologieal and Functional Status of Elements of the Reticuloendothelial System in Gnotobiotie Animals G. I. PODOPRIOORA, T. I. ZAITS~V, Research Laboratory for Experimental Biological Models, Moscow Region, USSR The reticuloendothelial system (RES) plays an important role in the organism's defense against infectious and other pathogenic agents. We studied the influence of the microbial factor on phagocytic activity and the morphology of elements of R E S of animals in gnotobiological experimental conditions. Conventional and germfree guinea-pigs (2 or 3 weeks old) as well as 3--4-month-old and 2.5-year-old gnotobiotic rats (3--4 months and 2.5 years old) were used in the experiments. Gnotobiotic rats were both germfree and tetrabiotie harboring triflora (Clostridium putrificus, Bacillus subtilis, Saccharomyces). We investigated the peculiarities of the blood clearance of E. coli 055 in guinea-pigs in vivo and the phagocytosis of Staphylococcus albus 9198 in peritoneal macrophages of rats in vitro. We also determined the percentage of Kupffer cells of the number of hepatocy~es, as well as the content of ribonucleoprotein, glycogen and fat in hepatocytes A decreased phagocytic activity of both pathogenic and nonpathogenic microorganisms in y o u n g gnotobionts was established. A dependence of phagocytosis of E. coli 055 on the presence of specific opsonins was shown. The digestion indexes of macrophages of 3 - - 4 - m o n t h conventional rats were several times higher than of the gnotobiotic ones. On the contrary, macrophages of 2.5-year gnotobiotic rats showed a 2--2.5 times higher S. albus digestion indexes than conventional ones (Table I). Histological studies established an increase of the number of Kupffer cells in the liver of b o t h conventional and

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TABLE I. Phagocytosis of S. albus b y peritoneal maerophagos of y o u n g and old gnotobiotie a n d conventional rats ( ~/oa) Time h b Animals

0.5

2.5

gnotobiotic

conventional

gnotobiotic

conventional

Young

(3-month)

6.0 • 2.5

57.5 -4- 9.8

16.2 • 5.0

83.8 • 5.3

Old

(2.5-year)

11.7 • 2.3

7.2 -4- 2.1

43.8 -]- 6.1

16.7 4- 7.1

The percentage of active macrophages -- phagocytes, in which more than one-half of ingested microbes am destroyed. b F r o m the=beginning of phagoeytosis.

germfree rats up to %he age of 90 d. The percentage of Kupffer cells among 1500 hepatocytes in gnotobiotic and conventional rats were, respectively: for 15-d-old animals -- 9.6 J: 0.4 and 18.5 -}- 0.8; for 30-d-old ones -- 15.2 -}- 0.07 and 24 :k 1.0; and for 90-d-old ones -- 17.2 -4- 0.8 and 83.3 -4- 1.4. The number of Kupffer cells in 300-d-old gnotobionts did not change significantly (16.3 ~ 0.7), while their number in conventional rats decreased (26.4 • 1.1). Thus the numbers of Kupffer cells in gnotobionts of all age groups were approximately twice lower than in conventional ones. A lower content of double-nuclear hepatocytes was found in gnotobionts of all age groups. A lower content of ribonueleoprotein granules and inconsiderable content of glycogen was observed in the cytoplasm of hepatocytes. A fat infiltration of hepatocytes and Kupffer cells was shown in adult gnotobionts. The established morphological peculiarities as well as decreased levels of opsonins explain the peculiarities of functional activity of R E S in young and adult gnotobionts. The accelerated decrease of phagocytic activity of I~ES in old conventional rats as compared with gnotobionts is connected with the general biological regularities of the aging process in conditions of noncontrolled microbial influence which is an important factor in limiting the life span of conventional animals.