Teresa I. Mercado

Observations on function, composition, and structure of cestode calcareous corpuscles

Abstract 1. 1. Taenia taeniaeformis contains about half as much calcareous corpuscle material as does Cysticercus fasciolaris. The corpuscles show no quantitative gradient along the strobila of Taenia taeniaeformis, but the larger individual proglottids contain more corpuscle material than the smaller ones. 2. 2. More calcareous material disappears from Cysticercus faciolaris during anaerobic than during aerobic incubation in non-nutritive media. In both cases considerably more corpuscle material disappears when the worms are incubated in slightly acid than in slightly alkaline surroundings. It is concluded that the corpuscles serve to buffer acids entering the body from the outside and, possibly, to buffer acids produced during aerobic and anaerobic fermentations. 3. 3. The corpuscles yield Ca, Mg, P, and CO2. The mineral component of the corpuscles is amorphous, but upon heating with KOH crystallization takes place and brucite and hydroxyapatite crystals are formed. The carbonates, however, do not crystallize. 4. 4. Electron microscopy indicates that the lamellae of the corpuscles may be paired rings. It reveals also an amorphous or granular background substance, but no crystalline elements. 5. 5. The inorganic material of the corpuscles can readily be demonstrated by various histochemical staining methods. It is also possible to demonstrate histochemically a glycogen-like polysaccharide, a mucopolysaccharide, lipids, and proteins as constituents of the organic base of the corpuscles.

Glycogen studies on white rats infected with Plasmodium berghei

Abstract The liver glycogen of female white rats infected with Plasmodium berghei decreased progressively with increasing parasitemia while a decrease in the carcass glycogen became apparent only in highly parasitized animals. Experiments with controlled food intake proved that the loss in carcass glycogen can be fully accounted for by the reduced food intake of parasitized animals, but this factor explains only part of the loss in liver glycogen. A true disturbance of the liver function has to be assumed in addition to the effect of semi-starvation. This view is substantiated by the observation that parasitized animals deposited less liver glycogen than nonparasitized controls when given glucose either orally or intravenously.

Trypanosoma cruzi: lactate dehydrogenase isoenzymes and infections in mice.

Abstract Total plasma LDH isoenzyme (EC 1.1.1.27) levels increased significantly over the normal level in mice infected with strains of Trypanosoma cruzi from three different geographic locations, but some strain differences were observed. The most rapid increase was exhibited by the blood-induced Tulahuen strain, but this strain, unlike the House 510 or House 11, did not elicit an increase during the early period of infection. Overall increases in LDH-1 and LDH-2, heart isoenzymes, were most marked in vector-derived House 510 infections, but, as in the Tulahuen strain, a considerable increase was also observed in blood-induced infections. The House 510 strain also elicited significant increases in LDH-4; these were particularly high during the early period of the blood-induced infection. By contrast, the vector-derived Tulahuen strain elicited a higher increase in LDH-4 during the early period than the House 510 or House 11 strains. Comparable similarites and differences were also observed in regard to LDH-3, 5, and “X.” The most marked isoenzyme increases were those of “LDH-X” exhibited by the blood-induced House 510 and vector-derived Tulahuen strains. Parallel histopathologic studies of liver, heart, and skeletal muscle disclosed significant pathology in all the infections. Animals with blood-induced Tulahuen strain infections characteristically showed extensive necrosis with marked multiplication of parasites throughout the liver, but little or no evident damage to the heart and skeleal muscle. Animals infected with House 510 and House 11 strains exhibited minimal pathology in the liver but severe damage to the heart and skeletal muscle. Increases in LDH-4 and LDH-5, isoenzymes which represent both liver and skeletal muscle, in blood-induced Tulahuen infections were attributed largely to liver damage, but in the House 510 and House 11 infections were related more to skeletal muscle.

Quantitative and histochemical studies on glycogenesis in the liver of rats infected with plasmodium berghei.

1. 1. Various hexoses and disaccharides, melezitose, and glycine led to the formation of liver glycogen in uninfected animals, but pentoses, d-glycero-d-guloheptose, and glutamic acid did not. Two other heptoses gave rise to only very small amounts. 2. 2. In all cases in which control animals showed a marked glycogen synthesis, the latter was less pronounced in infected animals. 3. 3. The relative glycogenic properties of carbohydrates given under comparable experimental conditions was in most cases similar in control and infected rats, only mannose and galactose, as well as the amino acid glycine, being relatively much less glycogenic. 4. 4. Fructose or glucose fed controls showed histochemically an even glycogen distribution in the liver, while in infected animals the polysaccharide was deposited almost exclusively in the cells adjacent to the portal vessels. 5. 5. The evaluation of the quantitative and histochemical observations lead to the conclusion that certain cells of infected livers can deposit and store maximum amounts of glycogen while others do not; whether formation, deposition, or both is disturbed in the latter could not be decided.

Histochemical localization of glycogen-synthesizing enzymes during parasitic infections

Abstract Polysaccharide synthesis by amylophosphorylase, glycogen synthetase, and branching enzyme was generally periportal in the liver of rats infected with Plasmodium berghei. In animals with low-grade infections synthesis was more uniform throughout the liver lobule resembling that of normal rats while in very highly infected animals (parasitemia over 70%) no significant activity was demonstrable. As in malaria, synthesis during hemobartonellosis was essentially periportal. In high trypanosome infections periportal synthesis was observed also but not as regularly as in malaria or hemobartonellosis; centrilobular activity was observed frequently. A periportal deposition of sugar corresponding to the areas of glycogen synthesis in the malaria-infected animals was not observed. In infected as in normal animals sugar appeared distributed more or less evenly throughout the liver lobule. The present findings parallel closely those obtained in previous in vivo studies on glycogenesis and indicate that a faulty enzyme mechanism is the factor responsible for the changes in glycogenesis during these infections.

Creatine phosphokinase isoenzymes and Trypanosoma cruzi infections.

1. Substantial increases in total creatine phosphokinase (CPK) and in isoenzymes from heart (CPK-MB) and skeletal muscle (CPK-MM) were observed during acute infections with the House 510 and House 11 strains of Trypanosoma cruzi. 2. In infections with the reticulotropic Tulahuen strain total CPK levels were lower and the isoenzyme pattern was essentially normal. 3. Gamma-glutamyl transpeptidase was considerably increased in the Tulahuen but not in the House 510 and House 11 infections. 4. These findings are useful in assessing tissue damage during T. cruzi infections and they also demonstrate differences between myotropic and reticulotropic strains which may aid in their taxonomic classification.

Plasmodium berghei: inhibition by splenectomy of a paralyzing syndrome in infected rats.

Abstract In recent attempts to isolate the factor causing paralysis in rats infected with a mousepassed KBG 173 strain of Plasmodium berghei Splenectomy was employed. The effects of Splenectomy on the paralyzing syndrome are discussed in the present report. Paralysis was inhibited in rats splenectomized prior to inoculation. Serial bloodpassaging of the strain in the splenectomized host however, apparently enhanced its virulence. Spleen-intact rats used as controls exhibited a marked increase in incidence of paralysis. Rats splenectomized a day before paralysis became evident were paralyzed significantly more frequently than those splenectomized a day earlier, indicating the apparent requirement of an incubation period for the expression of the paralytic effect. The enhanced virulence did not appear to be related to the level of parasitemia of the splenectomized rats used as donors. The spleen appears to provide the optimal conditions required for the elaboration of the paralyzing factor.

Metastatic Calcification induced by Hytakerol in Rats infected with Plasmodium berghei.

vitamin D, other derivatives of irradiated ergosterol, and parathyroid hormone induce metastatic calcifications in many organs (Vanderveer, 1931; von Brand et al., 1932; Engel, 1952; Baker et al., 1954; Haas et al., 1958). It is also known that several disease conditions, such as metastatic tumors, chronic renal disease, and hyperparathyroidism, are associated with abnormal tissue calcifications (Eisenstein et al., 1960). More rarely have studies involving parasites been reported. It has, however, been found that structures originating from the host, such as the wall of Trichinella cysts (von Brand et al., 1933, 1938; Wantland, 1934, 1935, 1936, 1938), readily calcify under the influence of the above compounds, as do tissues degenerating as a sequel of a parasitic invasion, such as the damaged tissue due to a larval tapeworm migrating through the liver (von Brand et al., 1933). Parasites, in general, seem liable to artificial calcification only after they have died (Otto and von Brand, 1941). Still less information is available concerning the possible influence of parasitic infections on the experimental production of metastatic calcifications in the tissues of the host. Von Brand and Holtz (1933) described an increased susceptibility to calcification of canary birds infected with Plasmodium praecox. It was considered worth while to examine this question further, employing a mammalian malaria species, Plasmodium berghei. In the present investigation, the effects produced by large amounts of a dihydrotachysterol concentrate on organ calcifications in the host were studied during the malarial infection in rats.

Hytakerol-Induced Metastatic Calcification in Trypanosomiases.

Heavy infections with Trypanosoma equiperdum and T. equinum reduced significantly the overall incidence of hytakerol-induced metastatic calcification, while infections with T. cruzi had no comparable effect. Differences between T. cruzi infections and infections with the African trypanosomes were also apparent in respect to calcification incidence of certain organs, e.g., the liver was definitely more calcified in the former than in the latter, while an opposite relationship was found in regard to the mosaic type of calcification of the stomach mucosa. No calcification around T. cruzi foci was found in any of the invaded tissues. Cold stress and cortisone administration increased rather than decreased the calcification susceptibility of uninfected rats. The question as to what role, if any, nonspecific stresses produced by the infection play in modifying the calcification response to hytakerol administration, cannot be decided unequivocally at present. In previous investigations of metastatic calcification induced by hytakerol in animals infected with malaria (Mercado and von Brand, 1962, 1964), significant differences were observed between two species of malaria, i.e., Plasmodium berghei in rats and P. gallinaceum in chickens. In earlier work von Brand and Holtz (1933) observed that the response of canaries infected with P. praecox to the calcifying effects of another ergosterol derivative, 7-dehydrocholesterol, also differed from that of the infected animals mentioned above. The differences involved mainly the overall incidence of calcification as compared to similarly treated control specimens, but differences in the susceptibilities of individual organs also occurred. Von Brand and Mercado (1956) and Mercado and von Brand (1960) reported that various parasitic infections such as malaria and trypanosomiasis induced different changes in liver glycogen and lipid. It was thought of interest, therefore, to extend the calcification studies to trypanosomiasis and to investigate whether or not the response of the host to the calcifying effect of hytakerol would be modified in this infection also. It was considered that such a comparative approach might shed light on the difficult question whether changes in physiological reactions in parasitized animals are due primarily to nonspecific stress produced by the presence of parasites or to specific influences from the parasites (e.g., toxins). Received for publication 18 September 1965. MATERIALS AND METHODS The United States Department of Agriculture strain of Trypanosoma equiperdum, the Wellcome strain of T. equinum, and the Tulahuen strain of T. cruzi were used. The T. equiperdum and T. equinum infections were developed in 6-week and 8-week-old Osborn-Mendel female rats, respectively. Six-week-old CFW mice were used for the T. cruzi infection. Generally 5,000 to 10,000 trypanosomes were inoculated intraperitoneally. Hemocytometer counting of the trypanosomes was done in glucose saline (1.6 g glucose in 800 ml normal saline, pH 7). This medium prolonged the survival of the flagellates and made counting possible for as long as 1 hr after withdrawal of the blood. Treatment with hytakerol (dihydrotachysterol concentrate, 20 X in sesame oil, with an activity equivalent to about 5 mg U.S.P. XVI dihydrotachysterol crystals per ml, Winthrop Laboratories) was initiated on the same day the rats were inoculated with T. equiperdum, 4 to 6 days after inoculation with T. equinum, and 6 days after the T. cruzi inoculations. The younger rats received one daily orally administered dose of 0.1 ml drug per 100 g body weight. The older rats and the 6-week-old mice received 0.08 ml and 0.3 ml, respectively. The drug was given for 4 days and the animals were killed on the 5th day. The main experimental series consisted of infected and uninfected animals which were treated with hytakerol, but a few shorter series consisting of uninfected and infected animals which did not receive hytakerol were completed also. In addition, two other series were done to test the possible influence of stress, induced by cold exposure and cortisone, on the calcifying effects of hytakerol in uninfected rats. In the first series rats were exposed to a temperature of 4 C for periods of 5, 10, and 17 days, corresponding controls being maintained at room temperature (22 C). Both groups were treated with hytakerol for 4 days as follows: The animals of the 5-day group received the drug starting on the 1st day of exposure and

Calcium in the liver of mice infected with Trypanosoma cruzi.

Biochemical and histochemical assays revealed that mice infected wtih the Tulahuen strain of Trypanosoma cruzi exhibit a significant enhancement of the level of liver calcium after 6 to 10 days of infection. In contrast to the changes observed in the liver the plasma level of calcium was normal. It is not possible to ascertain to what extent the amounts of calcium accumulated in the infected livers may have altered oxidative function in vivo; however, in vitro assays of normal livers revealed that incorporation of an amount of calcium equivalent to the increase observed in the infected livers did not influence the activity of cytochrome oxidase, an enzyme whose activity was reduced significantly during this infection. During the course of studies in this laboratory with Trypanosoma cruzi, it was observed that the activity of succinate dehydrogenase and cytochrome oxidase was reduced significantly in the livers of heavily infected mice (Mercado, 1969). This effect was attributed largely to the marked liver necrosis which accompanied the infection and consequently to a loss of mitochondria, the sites of these oxidative enzymes. In view, however, of the function of mitochondria in the regulation of cellular calcium (Vasington and Murphy, 1961, 1962; De Luca and Engstrom, 1961; Vasington, 1963; Lehninger et al., 1963; Rossi and Lehninger, 1964; Carafoli, 1967; Patriarca and Carafoli, 1968, and others), and because of the possibility that the reduced enzyme activity mentioned above may have been related to an accumulation of calcium comparable to that occurring in livers poisoned with thioacetamide or carbon tetrachloride (Gallagher et al., 1956; Reynolds et al., 1962; Reynolds, 1963, 1964; Reynolds and Yee, 1968), the present study was undertaken. MATERIALS AND METHODS The Tulahuen strain of Trypanosoma cruzi was used in 4-week-old CFW male mice inoculated intraperitoneally with 5 to 10 million trypanosomes. They were fed a diet of Purina chow and watered ad lib. They were killed by cervical dislocation. They were not perfused. The quantitative determination of calcium was made on ashed samples of a 10% liver homogenate prepared in double-distilled water. Three ml of the homogenate were placed in a weighed porcelain Received for publication 24 January 1972. crucible, reweighed, and allowed to dry at 100 C for at least 4 hr. The dry weight was taken after cooling in a desiccator, the samples were covered, and were placed in a muffle furnace set at 300 C. The temperature was raised to 600 C and ashing allowed to continue overnight. The following day the temperature was reduced to 300 C and the crucibles were removed, cooled, and weighed. One ml each of concentrated HC1 and double-distilled water were added to dissolve the ash. The deposits at the bottom and sides of the crucibles were carefully scraped into the acid solution with a glass rod. In addition the crucible cover was rinsed with acid in order to dissolve any ash that may have deposited on this surface. The acid solution containing the dissolved ash was then pipetted into a 10-ml volumetric flask containing 1 ml of a 10% lanthanum solution. The crucibles and covers were then washed 2 or 3 times with double-distilled water, the washings were transferred to the volumetric flask, and dilution completed to mark. After thorough mixing, the solutions were filtered through Whatman No. 1 paper, and portions assayed by atomic absorption spectroscopy. A Zeiss atomic absorption spectrophotometer employing an air acetylene flame was used. The light source was a Westinghouse calcium hollow cathode lamp. Samples of the experimental solutions were compared with calcium standards containing comparable concentrations of HC1 and lanthanum. The use of porcelain crucibles for dry-ashing was found satisfactory as long as adequate controls consisting of empty vessels, ashed, and treated as the experimental ones were maintained. Further check on accuracy of determinations was made by analyzing at least 2 samples of different concentrations of the homogenized material. Histochemical localization of calcium was studied on 7-g-thick sections of livers (one block from each main lobe) fixed in absolute alcohol, embedded in paraplast, and stained with the von Kossa and the alizarin red S techniques (McGhee-Russell, 1958). Control sections were treated with 5% acetic acid for 30 min.