Leishmania (Viannia) braziliensis: comparative pathology of golden hamsters infected with isolates from cutaneous and mucosal lesions of patients residing in Tres Bracos, Bahia, Brazil.

The histopathology of primary forepaw and metastatic lymph node, spleen, and liver lesions produced in golden hamsters infected with cutaneous leishmaniasis (CL) strains (LTB 111 and LTB558) and mucocutaneous leishmaniasis (MCL) strains (LTB12 and LTB201) of Leishmania (Viannia) braziliensis isolated from patients residing in Tres Bracos, Bahia, Brazil is described. No pathological features providing clear differentiation of the CL and MCL strains were found. Although amastigotes were plentiful early in the development of primary forepaw lesions, they were either absent or could not be identified with certainty in sections of late stage lesions. Similarly, amastigotes were not found in histologic lesions at metastatic sites; however, leishmanial DNA was detected in both early and late stage forepaw lesions and metastatic lesions using Leishmania kinetoplast DNA and the gene coding for gp63 as hybridization probes. The DNA recovered from metastatic lesions was extracted from formalin-fixed paraffin-embedded tissues that had been stored at room temperature for prolonged periods.

Pathology resulting from the administration of a live attenuated anti-Schistosoma haematobium vaccine in baboons.

Baboons (Papio anubis) were injected in the leg muscle with 18,000 20 Krad irradiated schistosomula of Schistosoma haematobium. Four protocols were followed: single, primary injection; single injection into animals primed by patent S. haematobium infection; secondary vaccine injection following an earlier injection; and single injection following praziquantel treatment of infected animals. Injection of the putative vaccine elicited localized mixed inflammatory infiltration at the site of injection which was both intense and prolonged. Three grades of tissue reaction were seen: the relatively mild primary response; the response in infected animals which had enhanced tissue eosinophilia; and the response in animals primed by prior injection and drug-treated prior infection. The latter 2 showed intensification of eosinophilia, stellate abscesses in the lesion centers, and perischistosomular Hoeppli precipitates. Intramuscular lesions peaked at 14 days for the primary response and at 7 days for all secondary responses. Traces of the milder lesions persisted beyond 4 weeks; more severe reactions healed more rapidly. Some schistosomula survived for 14 days in the milder reactions. A few larvae were deposited in the skin by backflushing of the injectate which produced local inflammation. Compared to mice, live schistosome vaccines injected into baboons elicited greater local inflammation; however, while evidence suggested that sporadic vaccine schistosomula did reach the lymphatic nodes draining the injection sites, no systemic lesions were found and the injection sites healed in approximately 5-6 weeks without permanent damage.

In vivo T cell depletion regulates resistance and morbidity in murine schistosomiasis.

These studies assessed the roles of subpopulations of T lymphocytes in inducing and modulating resistance to schistosomiasis and thereby influencing subsequent morbidity. C57BL/6 mice were depleted in vivo of Lyt-1+, Lyt-2+, and L3T4+ cells by the daily administration of monoclonal antibodies. The development of protective immunity, induced by exposure to irradiated Schistosoma mansoni cercariae as expressed in depleted animals, was compared to that demonstrated in undepleted, normal, and congenitally athymic C57BL/6 mice. The development of morbidity was determined by spleen weight, portal pressure and reticuloendothelial system activity. The results indicated that depletion of specific subpopulations of T lymphocytes minimally affected the primary development of parasites; however, depletion strongly influenced the development of resistance to the parasite and subsequent morbidity due to infection. Depletion of T lymphocytes by anti-Lyt-1+ or anti-L3T4+ antibody decreased the development of resistance, antibody and delayed-type hypersensitivity directed against schistosome antigens. Morbidity due to disease was increased. Depletion of Lyt-2+ cells produced opposite changes with augmented resistance and reduced morbidity. Congenitally athymic mice developed minimal resistance and morbidity. Moreover, resistance was inversely related to the morbidity shown by a given animal. These studies indicate that the development of protective immunity to S. mansoni cercariae is regulated by discrete subpopulations of T lymphocytes. The feasibility of decreasing morbidity by increasing specific immunologically mediated resistance is suggested.

Effects of protective immune serum on the yields of parasites and pulmonary cell reactions in schistosome-infected rats.

Yields of parasites during the period of worm migration from the lungs to the portal circulation were measured in S. mansoni-infected Fischer rats passively immunized with protective serum from twice-infected donor rats. Two effects of protective serum were observed in recipient rats relative to normal serum recipients: yields of schistosomula from lungs were higher and yields of (immature) worms from the portal circulation were lower throughout the period analyzed. Histopathological analysis of lung tissue confirmed the presence of greater numbers of schistosomula in lungs of passively immunized rats. In addition, the percent of lung schistosomula involved in all categories of inflammatory reactions was greater in recipients of protective rat serum. The kinetics of accumulation of worms perfused from the portal circulation of normal and passively immunized rats indicate that in the latter group a smaller fraction of worms successfully migrates to the portal circulation. These findings support the hypothesis that protective activity of the serum prevents a portion of worms from successfully completing migration from the lung to the portal circulation.

Immunopathology of Schistosoma japonicum infection in athymic mice.

Athymic (nu/nu) mice and heterozygous littermate controls (nu/+) were examined 7 and 10 weeks after infection with 10 cercariae of Schistosoma japonicum. Schistosome infection developed normally in both groups of mice and eggs were produced in normal numbers. Nu/nu mice developed small circumoval granulomas with minimal fibrosis while nu/+ mice developed large fibrotic granulomas. Unlike the mononuclear responses to S. mansoni eggs at 7 weeks, those to S. japonicum often were abscess like with narrow rims of liver cell necrosis or microvesicular fatty change. However, evolving granulomas in nu/+ mice were enriched with eosinophils, epithelioid macrophages, immature granulocytes and plasma cells, all scarce in the corresponding nu/nu lesions as were fibroblasts and collagen fibres, thus accounting for their smaller mean size and better healing. Our aggregate evidence shows that normal granuloma formation and cellularity in S. japonicum infection is controlled by T-cells as is the case for S. mansoni, and not by antibodies or immune complexes.

Immunopathology of Schistosoma japonicum and S. mansoni infection in B cell depleted mice.

To investigate the role of antibody in the pathogenesis of hepatic granulomas around schistosome eggs, mice were depleted of B cells by treatment from birth with anti-IgM serum and were subsequently infected with Schistosoma japonicum or S. mansoni. Anti-IgM treatment did not affect the development or fecundity of the worms or the larvae within the egg shells. Normal circumoval granulomas were present in the livers of B cell depleted mice 7 or 8 weeks after infection clearly indicating that antibody and immune complexes have no necessary role in the formation of granulomas. Hepatic fibrosis was also similar in B cell depleted and untreated mice at these times. Ten weeks after infection the size of S. japonicum egg granulomas in untreated mice had decreased but no change in the size of granulomas had occurred in B cell depleted mice, and hepatic fibrosis was more marked in treated than in untreated mice. Similar changes were noted in S. mansoni infected mice, assayed at 8 and at 12-13.5 weeks after infection. The effects of B cell depletion in the more chronic infections may be related to the absence of antibody but could also be caused by an influence on B cell-dependent suppressor T cells.

Pathology of a live attenuated anti-schistosome vaccine in mice.

Tissue responses of mice to intramuscular injection of 50 kR 60Co-attenuated schistosomula of Schistosoma mansoni were studied. Controls included injection of unattenuated schistosomula, medium alone, antigen-coated beads, and alum-adsorbed tetanus/diphtheria toxoids. Primary reactions to tissue-confined deposits of injected schistosomula, whether attenuated or not, were relatively intense and prolonged. Parasite attrition proceeded steadily, with most destroyed by the 7th day; however, a few intact organisms persisted up to 4 weeks. Cryopreservation did not alter the course of parasite attrition nor host reaction. Irradiated larvae were not found in lymph nodes, lungs, or liver. Neutrophils dominated the early reactions and were gradually replaced by mononuclear phagocytes, lymphoid cells, and eosinophils. Fibroblast proliferation and muscle regeneration began by day 3; reaction size and intensity peaked by day 7. From weeks 1-4, inflammatory infiltrates and regenerative proliferation underwent gradual involution, and injection sites were healed with no scarring by the end of 4-5 weeks. Mice primed by infection or by prior injection showed an accelerated course of inflammation, enhanced tissue eosinophilia, and more rapid healing. An unwanted, but prominent, feature of schistosomular vaccine reactions in mice was tracking of the inflammatory infiltrate along connective tissue septal and nerve sheaths, the latter raising the question of the pain potential of the vaccine. To conclude, in mice, attenuated schistosomular vaccines cause relatively marked local inflammatory responses but no systemic lesions at all, and their injection sites heal without permanent damage.

Hemoglobinolytic activity of serum in mice infected with Schistosoma mansoni.

Sera from mice infected with Schistosoma mansoni were found to contain substantial amounts of an acid-active hemoglobinolytic enzyme. Recovery of this enzyme from aliquots of pooled 12-week infected serum, using a phenylalanine-agarose affinity column, showed that a portion of the enzyme binds tightly to the column at pH 4.0, and can be eluted with 0.01 M formic acid. Another larger portion of hemoglobin-digesting activity is not bound to the column and emerges with the buffer front. Sera from rats which were exposed to cercariae, but in whom worms were stunted and did not develop to maturity, were found not to contain hemoglobin-active protease. At the present time, the source of the enzyme has not been unequivocably proven. The enzyme found in the serum binds to and releases from the affinity column in the same manner as does hemoglobinase recovered from freeze-dried S. mansoni worms. Maximal activity of both enzymes against the substrate occurs at pH 4.5-5.0. Present evidence suggests that the protease present in the serum is of worm origin. It is postulated that this protein may be excreted by the parasite during the process of regurgitation of gut contents. Presence of worm enzyme circulating in the host plasma would correlate with the known sensitization of the host to schistosomal hemoglobin-digesting enzyme.

Pulmonary cell reactions in natural and acquired host resistance to Schistosoma mansoni.

In order to examine the relationship between host leukocytic reactions to schistosomula and innate or acquired resistance to Schistosoma mansoni, in vivo pulmonary cell responses in CD/F rats, LVG hamsters, C57BL/6 and CBA mice, following either cercarial skin exposure or intravenous injection of schistosomula (the "lung model"), were quantified and analyzed. Major leukocytic reactions to schistosomula injected into the lungs varied according to host strain, with increasing responses occurring in the order CBA < LVG < C57 < CDF. Adult worm recoveries, by portal perfusion of these hosts, ranked in a strain order reciprocal to that of lung cell responses. All hosts developed anamnestic, eosinophil-enriched responses on secondary intravenous schistosomula challenge. In mice, this in vivo eosinophilic, augmented response could be elicited by glutaraldehyde-fixed as well as by intact challenge schistosomula. After primary percutaneous cercarial exposure, lung responses at 5 days were significant in rats, and after secondary challenge, in both rats and hamsters, but were virtaully nil in mice, whether previously exposed to S. mansoni or not. Thus, schistosomulum attrition was partly dependent on parasite encounters of various kinds with host mono- and granulocytes, but was of major consequence only in hosts with native (rat) or acquired resistance (all hosts), while playing a minor role in naive permissive hosts (mouse, hamster). The failure of previously-infected mice to develop early lung residual killing foci in response to skin-penetrated schistosomula is unique among the known laboratory hosts of S. mansoni.

Effect of Schistosoma mansoni infection on hepatic drug-metabolizing capacity of athymic nude mice.

The effects of infection with Schistosoma mansoni on the activities of several hepatic drug-metabolizing enzymes were investigated in congenitally athymic homozygotic nude mice and in a heterozygotic strain of BALB/c derived mice. In athymic nude mice, infection with schistosomes of the same duration and intensity (in terms of the number of eggs in the liver) as in heterozygotic mice resulted in a much smaller reduction in hepatic drug-metabolizing enzyme activities. Therefore, the severe reductions of the hepatic drug-metabolizing function in this infection occur only in mice that are immunologically competent and, thus, are dependent on the host's response to the parasite eggs.

Structure of the schistosome eggshell: amino acid analysis and incorporation of labelled amino acids.

Preparations of eggshells of Schistosoma mansoni and S. japonicum were hydrolyzed and analyzed for amino acid composition. Both species showed great similarities in the proportions of each residue found. The predominant amino acid in shell hydrolysates was found to be glycine, which accounted for 37% of S. mansoni and 45% of S. japonicum amino acids. Four components (glycine, aspartic acid, lysine, and serine) totalled 68--75% of amino acids in the eggshells. Other individual amino acids were present in relatively small proportions ranging from 5.2--0.01%. Less than 1% of the amino acid residues were identified as tyrosine, and bityrosine was detected at a level not exceeding 1 in 1,600 residues. Carbohydrates were estimated to comprise 7.5--10% of the eggshell weight, based on hexose assay, and glucosamine was identified as the principal amino sugar in shell hydrolysates. In vivo labelling of the S. mansoni eggshell was demonstrated following injection of C14-glycine and C14-tyrosine into infected mice and subsequent purification of the shells of eggs recovered from their liver.

Schistosoma mansoni infections in T-cell deprived mice, and the ameliorating effect of administering homologous chronic infection serum. II. Pathology.

Liver changes occurring in mice deprived of their T-cells by a combination of thymectomy and anti-mouse thymocyte serum, and in immunologically intact control mice, were followed during the early stages of heavy Schistosoma mansoni infections. Lesions in both groups began developing by day 38 and were maximal by day 48. Hepatic changes in control mice culminated in large hypersensitivity granulomas, tissue eosinophilia, portal periphlebitis, fibrosis, vascular obstruction, and infarction leading to arterialization and preferential sinusoidal channeling. Deprived mice showed greatly reduced egg reactions composed principally of macrophages, monocytes, and occasional neutrophils, and only minimal alteration of liver architecture; however, focal and disseminated hepatocellular lesions became prominent as the infections progressed, and by day 48 virtually every hepatocyte was affected. Typically, hepatocytes showed microvesicular cytoplasmic damage (steatosis) or ballooning degeneration with accompanying nuclear pyknosis or karyorrhexis. This cellular pathology may be attributed to the direct or indirect effect of eggs or egg products on liver cells. The administration of chronic infection serum obtained from immunocompetent mice to T-cell deprived mice dramatically eliminated the hepatocellular lesions. It also increased eosinophil participation and fibrosis in the egg reactions but did not restore the size and other cellular features typical of egg hypersensitivity granulomas. Serum from uninfected normal mice was found to lack these effects.

Potentiation of schistosome granuloma formation. By lentinan--a T-cell adjuvant.

Lentinan is a fungal polysaccharide which acts as a T-cell adjuvant. When this glucan was administered to thymus-intact mice by intraperitoneal injection, conspicuously enlarged lung granulomas formed in response to either Schistosoma mansoni or S japonicum eggs or to antigen-coated polyacrylamide beads. Liver granulomas in cercaria-induced S mansoni infection were augmented up to eight-fold in volume. By contrast, nude mice showed a complete absence of hypersensitivity granulomas, regardless of whether they received lentinan. Lentinan-potentiated granulomas show a distinctive histopathologic picture characterized by abundant, large, pale-staining macrophages; reduced and redistributed eosinophil populations; and frequent, extensive central necrosis, uncommon in unpotentiated schistosome foci. They also differ in their distributions of egg antigen and of host immunoglobulins. Optimal lentinan effects followed a single 1-mg dose when given to sensitized mice on the day of intravenous challenge with S mansoni eggs rather than at the time of intraperitoneal sensitization or following challenge. This adjuvant appears to act on effector T cells or on macrophages interacting with T cells; its effect on macrophages in a latex bead foreign body granuloma was minimal. A number of other lentinan-associated systemic effects on parasite and host were noted and described, including reduced female schistosome egg output.

Tissue eosinophil proliferation and maturation in schistosome-infected mice and hamsters.

Extramedullary eosinophilopoiesis is described as a regular pathological feature of murine and cricetine schistosome infections. Using the Dominici staining technique, colonies of developing eosinophils were commonly found in certain tissue sites of dense oviposition, particularly in the liver, and in associated lymphoreticular tissues. The relationship of these observations to the role of eosinophilic leukocytes in schistosomiasis is discussed.

Altered schistosome granuloma formation in nude mice.

Schistosome egg-induced lesions in congenitally athymic mice differed from those found in normal heterozygous controls. Heterozygote liver granulomas were chareacterized by poorly phagocytic epithelioid macrophages, and were rich in eosinophils and fibroblasts, with peripheral lymphocytes and plasma cells. Hepatic lesions in nude mice were much smaller and lacked epithelioid macrophages, with lesions about mature eggs, typically consisting of monocytes and macrophages filled with pigment, occasional neutrophils, and rarely one or more eosinophils or giant cells. While heterozygote granulomas damaged liver cells mainly by encroachment or by their vascular effects, in the nudes hepatocytes bordering the lesions showed microvesicular cytoplasmic damage and either hydropic degeneration or focal acidophilic necrosis of individual liver cells. In heterozygotes, immunofluorescent-stainable schistosome egg antigen (SEA) was concentrated in the granuloma center. In nude mice, SEA, was distributed throughout the infiltrates and in and around hepatocytes adjacent to egg lesions corresponding to the observed pattern of hepatocyte necrosis. We conclude that, in contrast to heterozygotes, nude mice lack hypersensitivity granulomas and fail to sequester toxic egg products, this resulting in zonal hepatocellular damage. Alternative explanations include the possibility of a latent hepatitis virus being activated by the schistosome infection; however, several cogent arguments are presented against that alternative.

The structure of the ovarian ball and oogenesis in Moniliformis dubius (Acanthocephala).

Seven to nine days after infection of the definitive host (rat) by cystacanths, the genital primordium of the female acanthocephalan is transformed from a fragmented mass of cells into discrete ovarian balls. This is accomplished by envelopment of free germinal cells by somatic tissue which originates from the ligament sac primordium. Germinal cell nuclei then undergo repeated mitoses until about 21 days of development, with concurrent formation of oogonial syncytia which occupy the interior of the ovarian balls. Oocytes, derived from these oogonia, move to the periphery of the germinal syncytia for differentiation, growth, fertilization, shell formation, and release from the ovarian ball. After oogonial proliferation ceases, continued growth of the ovarian ball apparently results from increase in size of already present cells. Free-floating mature ovarian balls are found in the dorsal ligament sac; each consists of germ cells in various developmental stages, enveloped and pervaded by a multinucleate matrix syncytium of somatic origin, which functions as a follicle. Spermatozoa pass through the matrix cell for the internal fertilization of mature oocytes. Myelinated structures of an undetermined nature were found to correspond to previously reported polar bodies. After 100 days post-infection, the somatic matrix syncytium begins to manifest the degenerative effects of aging. The germinal tissue exhibits no subcellular signs of senescence by 154 days, but decreases in amount in older worms.

Viruslike inclusions in the cecal epithelial cells of Pa agonimus kellicotti (Digenea, Troglotrematidae).

Rodlike or tubular inclusions are described from the cytoplasm and nuclear matrix of the cecal epithelial cells of Paragonimus kellicotti. These inclusions are 3.4 mu or more long, 350 A in diameter, and comprised of a wall of helically arranged subunits and a dense, possibly filar, central core. They occur in organized bundles of up to 17 members. Cells containing these inclusions manifest a series of nucleolar modifications typical of inclusions is discussed, noting especially their resemblance to known rodlike viruses from other systems.