José Oliver-González

Effect of Hemolymph from Biomphalaria glabrata on Schistosoma mansoni Infection in Mice

Discussion and Summary Recent studies have indicated that when foreign blood components are injected into mice infected with S. mansoni, the metabolism of the worms is affected resulting in changes in oviposition and death. Intraperitoneal injection of washed erythrocytes from Macaca mulatta injected into mice resulted in diminution of egg laying capacity and death of females (2). Also injection of Forssman antigen, prepared from guinea pig kidney resulted in an increased pigment formation in the worms and eggs laid in the tissues (3). Injection of hemoglobin from nonmammalian sources, and thus very foreign to mice, was thought of as a good prospect, with the idea that the metabolism of the worm could be altered more efficiently. Michelson (4) reported that the hemolymph from B. glabrata has at least four major hemoglobin components, thus the hemolymph from this snail was used since they are easily available. Injections of snail hemolymph into infected mice resulted first in changes in the oogram with the disappearance of immature eggs indicating a rapid effect on the females, and finally death of both sexes. After death of the worms, the eggs remaining in the tissues were rapidly absorbed, and the liver returned to normal gross and microscopic contexture. The mechanism by which the worms are killed is not totally understood. The hemocyanins and hemolymphs are markedly immunogenic substances, which makes it easy to postulate that death of the worms is caused by an immunological reaction between antibodies in mouse plasma and hemolymph antigen in the intestinal ceca of worms. Since mice developed high titered antibodies against the hemolymph, as shown by the agar gel reactions in which a 1:64 dilution of hemolymph was still positive, the antigen-antibody reaction could take place between the antibodies in plasma against homologous or heterologous hemoglobins within the worm. Such reaction might cause disturbances in the metabolism of pigment which deposits in large amounts and is not properly disposed. The marked accumulation of pigment observed in the dead worms suggests that this may be the case (Fig. 1). It could also be speculated that the snail hemolymph or its degradation products become toxic to the worm, without postulating an immunological reaction. This, however, is unlikely since the snail serves as the intermediate host for the parasite, in which development takes successfully. Changes observed in the livers of infected mice injected with snail hemolymph are very significant. The tissue returns to a normal histological structure and gross appearance. These observations are being investigated in more detail.

Adsorption of α2isoagglutinogen-like Substances of Infectious Agents on Human Erythrocytes.

Summary 1. Partially purified polysaccharides isolated from dry worm material from adult Ascaris lumbricoides (var suum); the larval forms of Trichinella spiralis and adult Taenia saginata adsorbed onto human erythrocytes of groups O and B, when these were incubated at 37°C or stored at 6°C in saline suspensions of the polysaccharide. 2. The treated erythrocytes were added to human serums of groups O and B and the α2 isoagglutinin titers were reduced to zero. No significant reduction of the α2 isoagglutinin titer was observed when control erythrocytes were added to the serums. 3. The titers of the α1 was slightly reduced and that of the β isoagglutinins was unaffected. 4. The fact that the erythrocytes treated with the polysaccharides inhibited the α2 agglutinins is used as evidence to show that the polysaccharides adsorbed onto the red cells. 5. Adsorption of polysaccharides with α2 isoagglutinogen-like properties, onto erythrocytes of human groups O and B gives the cell characteristics pertaining to a different group, i.e., the α2 group. The possibility of such phenomenon occurring during actual infection, and the relationship which it may have with the development of auto-hemagglutinins is mentioned.

Intradermal Response to Egg Antigen in Humans with Active and Treated (Fuadin) Schistosoma mansoni Infections

Summary 104 Puerto Rican individuals known to be infected with Schistosoma man-soni were skin tested with antigen prepared from lyophilized eggs and cercariae. 35.6% reacted to egg antigen while 95.2% reacted to the cercarial antigen. The discrepancy was found to lie in those who were passing living eggs and had no skin sensitivity to egg antigen while those who had been treated more than a year before approached in percentage the skin reactivity with cercarial antigen.

Blood Agglutinins in Blackwater Fever.

The serums from two cases of blackwater fever were tested for content of α, β, and cold agglutinins.∗ To 2 drops of the serum dilutions one drop of a 2% suspension of Groups A, B, and O erythrocytes was added. The suspensions in Wassermann tubes were incubated in a water bath at 37°C for 30 minutes followed by storage in the icebox at 6°C overnight. Examinations of suspensions for the presence of agglutinated erythrocytes was done after exposure to 37°C and 6°C with the aid of a dissecting microscope at a magnification of X20. Since both serums contained α and β agglutinins, it was necessary to absorb these in order to test for cold agglutinins, Small portions of serums were treated three times with one-half volume of packed fresh Group AB erythrocytes. During each absorption, the mixture was incubated at 37°C for 30 minutes, then centrifuged and the supernatant was treated again with fresh Group AB erythrocytes. Small portions of serum were also treated with a polysaccharide isolated from Ascaris summ which has been shown to inhibit specifically the α and β agglutinins in human serums. 1 enough of the polysaccharide was added to make a 4% suspension. The mixture was incubated at 37°C for 30 minutes and then tested. The serums treated with the Group AB erythrocytes and with the ascarid polysaccharide, as well as the untreated control serums, were then tested with Group A, B, and O erythrocytes and examined after exposures to 37°C for 30 minutes and at 6°C overnight. Table I shows that the serums from the 2 cases of blackwater fever had markedly increased α isoagglutinin titers of 1:8112 and 1:2028, respectively. After treatment of the serums 3 times with the Group AB erythrocytes, as well as after a single treatment with the ascarid polysaccharide, no α, β, or anti-0 agglutinins were detected after incubation at 37°C.

Treatment of experimental intestinal trichinosis with 1-diethylcarbamyl-4-methylpiperazine hydrochloride (hetrazan).

The drug l-Diethylcarbamyl-4-methylpi-perazine Hydrochloride (Hetrazan), when administered orally, causes the rapid disappearance of the microfilariae of Wuchereria bancrojti from the blood of infected individuals. 1 The marked effect of this drug on the circulating forms of W. bancrojti suggested its use in other parasitic infections in which treatment should be directed against such migrating forms. In view of the need for a drug in trichinosis which would destroy the migrating larvae as well as the intestinal forms, Hetrazan was tested on white rats infected with Trkhinella spiralis. The effect of this drug on the intestinal trichinae is reported below. Methods and Results. White rats weighing from 150 to 175 g were fed by stomach tube with 1,000 to 1,300 infective trichinae larvae. The larvae were obtained from rat muscle digested in a pepsin-hydrochloric acid mixture. Twenty-four hours after feeding the animals were started on Hetrazan. The drug was given on the basis of 200 mg per kg weight 3 times daily by stomach tube, for periods of 5 to 10 days. For the recovery of adult trichinae the treated animals and suitable controls were killed 24 hours after the last dose of the drug was administered. The small and large intestine were removed, slit open and cut into small pieces about 1 cm long. The sliced intestine was put in a Baermann apparatus with normal salt solution heated to 37°C. The adult worms settled rapidly to the tip of the funnel and were removed one hour after the apparatus was set up. The number of worms recovered from the intestines was determined directly by counting. For the recovery of the muscle stages the rats were killed on or about the 30th day after feeding infective larvae. The rat muscles were ground and digested in a pepsinhydrochloric acid mixture. The larvae were recovered from the mixture and their number estimated by counting aliquot portions of the total suspension.

In vivo Inhibition of Blood Agglutinins by Polysaccharides from Animal Parasites.

Summary 1. Monkeys and rabbits were injected intravenously with various amounts of polysaccharides (dissolved in 0.85% saline) prepared from the muscle stage of Trichinella spiralis and from adult Ascaris lumbricoides from pig. 2. The titer of the α2 isoagglutinins detected at 37 °C in the serums of the animals was reduced to negative for a period of 24 hours, and remained lower than the titer before inoculation for a period of 96 hours. 3. The titer of the α1 was also reduced, but not as much as the α2 agglutinins. 4. The titer of the agglutinins acting at 6°C against A1, A2 human, sheep, and the animals own erythrocytes was also reduced after inoculation of the polysaccharide. 5. The possibility of injecting these polysaccharides to inhibit autoagglutination of erythrocytes as seen in certain pathological conditions is mentioned.

Agglutination of erythrocytes in vivo in mice after injection of Ascaris extracts, related to immunization with human erythrocytes.

Summary Mice of an A strain were injected intraperitoneally with suspensions of human erythrocytes of Groups B and O, and of subgroups A1 and A2. Ten to 15 days after last injection of erythrocytes the animals and normal controls were injected intravenously with 1 mg of an extract prepared from cuticle of Ascaris lumbricoides. The purpose of immunization with erythrocytes was to determine whether the α1, α2, β, and anti-O agglutinins, developing as a result of artificial immunization, would react in vivo with cuticular extract. Histopathological examination of livers and kidneys disclosed: 1. Agglutination of erythrocytes with formation of large clumps in liver and kidneys was seen only in animals immunized against A2 erythrocytes followed by the cuticular extract. This did not occur in mice inoculated with erythrocytes of Groups B and O and subgroup A1, also injected with the cuticular extract. No such changes were seen in liver and kidneys of normal mice inoculated with A2 erythrocytes or cuticular extract alone. Agglutination takes place, therefore, between α2 agglutinins in plasma and the A2 isoagglutinogen-like factor in the extract. These findings indicate a definite relationship between blood groups and pathological changes produced by infectious organisms.

Treatment of Schistosomiasis mansoni with Neostibosan.

Twelve patients with ova of 5. mansoni in their stools were treated with the pentavalent antimonial drug, neostibosan (Winthrop). The patients were all males with ages ranging from 15 to 32 years. The drug was administered intravenously during a 2-week period of hospitalization. During the first 3 days, doses of 0.2, 0.6 and 0.9 respectively were usually given, and thereafter either 0.6 or 0.9 was generally injected every day until the end of hospitalization. All patients tolerated the drug well and no serious reactions were seen. A low grade fever and abdominal pain, headache, nausea or anorexia were observed in most of the patients. The effect of the drug on the number of schistosome eggs passed in stools was carefully observed. Stool examinations were made daily before and during treatment and on as many daily specimens as possible for each follow-up examination. The technic used for the quantitative determination of eggs in feces has been described previously. 1 The effect of treatment on the schistosome J infections, as determined by the number of alive and dead schistosome ova in stools may be seen in Table I. Although the majority of the patients (9 out of 12 or 75%) had stools negative for live and dead ova one to 2 months after treatment, 8 out of 11 (73%) relapsed 3 and 4 months later. Three out of 11 patients, however, have remained negative throughout, for a period of 11 months. Although the per cent of cases which has remained negative for ova of S. mansoni after treatment with neostibosan is small (3 out of 11 examined or 27.3%) there is evidence indicating that the drug has parasitotropic effects. This is also suggested by the fact than the egg count 11 months after treatment in 6 of the patients are appreciably lower than the counts before treatment. There is the possibility that in those patients in whom the infection was not eradicated insufficient drug was administered.