Simultaneous occurrence of hepatic alveolar and cystic echinococcosis.

The co-occurrence of hepatic cystic echinococcosis (CE) and alveolar echinococcosis (AE) is extremely rare. Here, we present the clinical manifestations and treatment outcomes of three cases with co-occurring CE and AE in the liver. Computed tomography (CT), magnetic resonance imaging and 18FFluorodeoxyglucose Positron Emission Tomography-CT were used for preoperative diagnosis. Specimens were taken intraoperatively and sent for pathological studies to confirm the coexistence of CE and AE by laminated membrane, daughter cysts or germinal layer and infiltration structure. Albendazole was prescribed after operation for 12 months. All patients were completely recovered and showed no recurrence at last follow-up. Therefore, surgical intervention and postoperative application of albendazole are recommended for patients with concurrence of hepatic AE and CE.

Surgical treatment of hepatic cystic echinococcosis in patients co-infected with HIV/AIDS.

Co-infections of cystic echinococcosis (CE) and HIV/AIDS is rare. We report four CE cases that were HIV positive. Three out of the four patients underwent a surgical operation to remove the hydatid cysts in their livers. The operation confirmed that in two of the cases their cysts had ruptured. These patients were given 3 months of albendazole after the operation. Follow-up showed they were remarkably improved in term of their health, although they were still HIV antibody positive 6 months after surgical treatment. Interestingly, the treatment remarkably increased their CD4+ cell population. We showed that surgery is suitable for treating hepatic cystic echinococcosis with HIV/AIDS co-infection.

[A case of pulmonary dirofilariasis].

A 76-year-old woman admitted to our hospital with abnormal shadow on chest X-ray. Computed tomography demonstrated the 2 x 2 cm of round, well defined tumor on right S6 segment which was suspected to be a malignant tumor. Partial resection was performed with video-assisted thoracotomy (VATS) and histological examination of quickly frozen specimen showed no malignancy. Then, histological examination of formalin fixed specimen showed dirofilariasis. It has been reported 97 cases of dirofilariasis in Japan so far. Although X-ray or biochemical and immunological examination of serum help to diagnose dirofilariasis, difinite diagnosis can be done only by histological evidence of resected specimen. Because of the difficulty for denial the malignant tumor, surgical treatment should be required.

Characterization of an immunogenic glycocalyx on the surfaces of Cryptosporidium parvum oocysts and sporozoites.

Ruthenium red staining of Cryptosporidium parvum oocysts revealed the presence of a carbohydrate matrix on their outer bilayers that is characteristic of a glycocalyx. Surface labeling of intact oocysts identified material of high molecular weight (>10(6)) that reacted positively with sera from cryptosporidium-infected patients and with immunoglobulin A monoclonal antibodies.

Leishmania donovani: pilot study for evaluation of therapeutic effects of inosine analogs against amastigotes in vitro and in vivo.

The inhibition of carbocyclic inosine (C-Ino), 3'-deoxyinosine (3'-dI), and 3'-fluoroinosine (3'-FI) to Leishmania donovani amastigotes was examined. J774.1 cells (a mouse macrophage line) were cultured in GIT medium with lipopolysaccharide and hemin and infected with the parasite. C-Ino (3 microM) completely inhibited and 3'-dI (30 microM) reduced to 40% the infection rate on Day 6 after infection. Pentostam (30 microM) resulted in a 38% infection rate. The therapeutic efficacies of nonentrapped free and liposome-entrapped inosine analogs were tested in mice infected with L. donovani. The mice were injected intravenously five times on alternate days, beginning 2 days after infection. Treatment with the nonentrapped free inosine analog of C-Ino (100 mg/kg), 3'-dI (100 mg/kg), or 3'-FI (50 mg/kg) resulted in an LDU that was 94, 68, or 73% lower, respectively, than the control values. Treatment with the corresponding entrapped inosine analog (10 mg/kg) caused decreases of 90, 69, or 68% LDU, respectively. The entrapped inosine analogs were inhibitory at doses one-fifth to one-tenth of the nonentrapped free inosine analogs. C-Ino had the strongest inhibitory effect among the three analogs tested in vitro and in vivo. Liposome-entrapped C-Ino had no severe side effects, although spleen weight increased. The agent may be useful as an anti-leishmanial drug.

Rejection of parental meth-a tumor on concomitant inoculation of meth-a cells infected retrovirally with the interferon-gamma gene into (balb/cxc57bl/6)f-1 mice.

The IFN-gamma gene was introduced retrovirally into Meth A cells. IFN-gamma gene infected Meth A (K gamma) cells were highly antigenic and regressed in CB6F(1) mice. Concomitant immunization of CB6F(1), mice with IFN-gamma gene infected Meth A (K gamma) cells after inoculation of parental Meth A protected the mice from parental tumor growth. 1x10(6) infectant Meth A (K gamma) cells protected the mice from growth of 1x10(6) parental Meth A cells, but 2x10(6) infectant cells did not, suggesting that there was an optimal dose of infectant cells for rejection of the parental tumor. Specificity analysis revealed that growth of CMS13 tumor was slightly inhibited by Meth A (K gamma) cells but that of CMS5 was not inhibited. The findings are consistent to those obtained with parental Meth A cells and indicated that the relevant rejection antigen on Meth A (K gamma) cells was identical to the parental Meth A rejection antigen.

Retention of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in the liver of mice infected with Schistosoma japonicum.

Regarding the mechanism underlying the suspected enhancement of hepatic cancers among Schistosoma japonicum-infected humans, we hypothesized that mutagen exposures in the livers of patients may be enhanced due to the parasitic infection. To explore this possibility, we have done a model experiment using mice and a carcinogenic mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Mice infected with Schistosoma japonicum were intravenously administered Trp-P-2, and the mutagenic activities of the mouse serum and of the liver tissue extracts, which were observable during the 6-h period after the administration, were investigated. The level of serum indirect mutagenicity, which probably reflected the amount of unmetabolized Trp-P-2, was higher in the infected animals than in uninfected control animals. Direct mutagenicity in the serum, on the other hand, was higher in the control animals than in the infected mice. Furthermore, the liver tissue extracts from infected mice showed higher indirect-mutagenicity than those from the controls. These data suggest that the infection results in a decreased metabolism and an increased retention of Trp-P-2 in the liver. Consistent with this phenomenon, pigments in the liver formed by the schistosome infection were found to be an efficient adsorbent for Trp-P-2. Thus, the possibility exists that these pigments, which contain hematin as a major constituent, may function as a reservoir for the mutagen, thereby prolonging the exposure period of the liver to the mutagen.

Parasite infection and cancer: with special emphasis on Schistosoma japonicum infections (Trematoda). A review.

This article contains a review of current knowledge on the association of parasite infections and cancer formation, especially that of Schistosoma japonicum (Trematoda) in man and experimental animals. The association of S. haematobium infection and bladder cancer is well known and documented. However, S. japonicum infection has also been reported to be associated with cancer, in this case hepatocellular carcinoma and/or colorectal cancer. Pathological records and analyses have shown a correlation between this infection and cancer, and pathohistological descriptions have been numerous, together with clinical case reports. Epidemiological analyses have been conducted in China and Japan and support a role of S. japonicum infection as one of the risk factors in cancer formation, along with others, such as hepatitis virus infection and alcoholic intake. Experimental results have also shown that cancer appears early and in larger numbers in experimentally infected animals given a known carcinogen. In spite of these positive end-point associations, the mechanism of schistosome-mediated enhancement of carcinogenesis is obscure. A suggestive observation is that in S. japonicum-infected mice carcinogen-metabolizing hepatic activity including P-450 was decreased so that an administered carcinogen persisted for a longer period than in uninfected mice. Further studies, both epidemiological and experimental, are needed to firmly establish the relationship between schistosome infection and cancer.

Modified metabolism of a carcinogen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by liver S9 from Schistosoma japonicum-infected mice.

Schistosoma japonicum infection has been associated with an increased incidence of liver and colorectal cancers in humans. To explore the mechanisms underlying this association, we investigated the carcinogen-metabolizing properties of liver S9 preparations from S. japonicum-infected mice and compared them with those of S9 from uninfected animals. When the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was incubated with these S9s and the products were analyzed by high-performance liquid chromatography, we observed that the S9 from infected mice had a lower ability to convert Trp-P-2 into 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), an activated form of promutagenic Trp-P-2, than the S9 from uninfected mice. We found that both of these S9 preparations have a high ability to reduce Trp-P-2(NHOH) into Trp-P-2; however, the infected-mouse S9 showed a significantly greater reducing power than the control S9. This difference appears to be responsible for the observed lower mutagen-activating potential of the infected mouse S9. These results suggest that hepatic enzyme activities of S. japonicum-infected mice are quantitatively different from those of normal mice.

Ultrastructural study of asexual development of Cryptosporidium parvum in a human intestinal cell line.

The lack of a well-defined in vitro model of Cryptosporidium infection has severely hampered research on the biology of parasitic invasion of the host cell and on intracellular development of the parasite. In vitro infection of the differentiated human enterocyte cell line HT29.74 was studied by electron microscopy to detect changes in parasite and host cell morphology. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned differentiated HT29.74 cells. Parasites and infected cells were evaluated by transmission electron microscopy at 20 min, 1 h, 6 h, 24 h and 7 days. Sporozoite invagination within the epithelial cell microvilli and subsequent penetration was evident at 1 h. At 6 h, the development of a dense band and feeder layer was visible. Development of the trophozoite into a schizont occurred over 24 h. Micronemes and dense granules were clearly visible within sporozoites and merozoites. Organization of vacuoles within the cytoplasm of the host cell was evident below the dense band. A sexual Cryptosporidium development in vitro was morphologically no different from initial development in vivo. In vitro infection of HT29.74 cells provides an excellent model to study parasite-host cell interaction and asexual parasite development.

Trypanosoma cruzi: localization of neuraminidase on the surface of trypomastigotes.

Immunoelectron microscopy (IEM) using TCN-2, a monoclonal antibody specific for Trypanosoma cruzi neuraminidase (NA), was performed to determine the precise localization of the parasite enzyme. In agreement with previous observations, TCN-2 reacted with tissue culture trypomastigotes, but not with epimastigotes, amastigotes or intracellular forms in intermediate stages of development. NA was localized on the surface of tissue culture trypomastigotes and in the Golgi apparatus suggesting that the enzyme is modified post-translationally. In agreement with this suggestion, digestion of NA with N-Glycanase, an enzyme that releases N-linked oligosaccharides, decreased the molecular weight of the polypeptides that make up NA.

Selective association of a fragment of the knob protein with spectrin, actin and the red cell membrane.

The knob protein of Plasmodium falciparum is essential for the formation of knob-like protrusions on the host erythrocyte membrane. A functional domain of the knob protein was identified. This peptide formed stable complexes with the two major red cell skeletal proteins, spectrin and actin. When introduced into resealed normal erythrocytes, the peptide associated selectively with the cytoplasmic surface of the membrane and formed knob-like electron dense deposits. Knobs are thought to play an important role in the immunopathology of P. falciparum infections. Our findings provide a first step towards understanding the molecular basis for selective membrane changes at knobs.

Asexual development of Cryptosporidium parvum within a differentiated human enterocyte cell line.

Unremitting diarrhea with malabsorption is associated with Cryptosporidium parvum infection of the small intestine in patients with AIDS. The lack of a well-defined in vitro model of C. parvum infection has severely hampered research into the biology of cryptosporidial invasion of the host epithelial cell and development of new pharmacologic and immunologic therapies. The adherent human intestinal epithelial cell line HT29 when grown in glucose-free medium develops morphologic and functional characteristics of the small intestine enterocyte and was used to develop an in vitro model of infection. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned, differentiated HT29.74 cells. Cells were fixed and stained to estimate the degree of parasite infection. Schizonts were easily distinguished from the host cell by light microscopy. Twenty-four hours after 10(5) oocysts were added to approximately 10(6) HT29.74 cells, Cryptosporidium infection rates varied from 50 to 120 schizonts per 1,000 cells. Among 14 different experiments, the mean infection rate was 91 (+/- 18) schizonts per 1,000 cells. Electron microscopy at 6 and 24 h confirmed intracellular localization and development of schizonts. The morphologic features of the cryptosporidial schizonts within HT29.74 cells, which included the presence of a dense band and feeder layer, were identical to those described during cryptosporidial infection of human enterocytes in patients with AIDS. Fewer schizonts were observed at 5 days and beyond. Infection of differentiated HT29.74 cells (62 and 65 schizonts per 1,000 cells at 24 and 72 h, respectively) was over five times more efficient than infection of undifferentiated HT29.74 cells (9 and 5 schizonts per 1,000 cells at 24 and 72 h, respectively). In vitro infection of differentiated HT29.74 cells will allow a better understanding of the mechanisms by which C. parvum infects the small intestinal epithelium and will allow a systematic evaluation of new therapeutic agents.

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

Reduced levels of mutagen processing potential in the Schistosoma japonicum-infected mouse liver.

Epidemiological studies have shown the presence of a positive correlation between the infection of Schistosoma japonicum and colorectal and/or liver cancers in the humans. To explore the mechanism underlying this correlation, we have investigated the mutagen-activating potentials of the liver homogenate fraction (S9) from Schistosoma japonicum infected mice and those from control mice, by use of the Ames test with 2-acetylaminofluorene, aflatoxin B1 and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) as test mutagens. Liver S9 prepared from the infected group at the 15th week after the infection showed a potential significantly lower than that from the control group. The hepatic cytochrome P-450 concentration in the infected mice was persistently low, about a half of that in the uninfected mice, during the period of 6-18 weeks after the infection. Thus, in mice bearing chronic schistosomiasis, mutagen-processing potentials are decreased.

Evaluation of the mutagenicity and the tumor-promoting activity of parasite extracts: Schistosoma japonicum and Clonorchis sinensis.

In relation to the observed association of carcinogenesis with parasitic infections, the mutagenicity of extracts of Schistosoma japonicum and Clonorchis sinensis was examined. In the bacterial mutagenicity tests using the Ames Salmonella typhimurium strains TA98, TA100, TA97 and TA102, and Escherichia coli WP2 and WP2 uvrA pKM101 Schistosoma soluble egg antigen and a homogenate of adult Schistosoma worms showed no positive responses either in the presence or in the absence of S9 mix. Likewise, adult worm extracts of Clonorchis showed no mutagenicity. The Schistosoma soluble egg antigen showed a weak but significant activity for the induction of Epstein-Barr virus expression in viral genome-carrying human lymphoblastoid cells in culture. This phenomenon suggests that the soluble egg antigen possesses tumor-promoting activity.

[Transition of occurrence of anisakiasis and its paratenic host fishes in Japan, with pathogenesis of anisakiasis].

The number of anisakiasis according to a nation-wide statistical survey totaled 4682 cases until June 1987 in Japan. Of these 4296 cases are gastric anisakiasis (including 215 cases of gastric terranovasis), 375 intestinal anisakiasis and 11 extra-gastrointestinal anisakiasis. Pseudoterranova larva does not invade the intestine, and worms are vomited in most cases. Clinical diagnosis of intestinal anisakiasis is more difficult than that of gastric anisakiasis, and it also is hard to find the worm itself in histopathological examination. Therefore the number of actual intestinal anisakiasis is probably 3 times more than that of the reported cases. The cases of gastrointestinal-wall perforation by worms are increasing, which means immune response by granuloma formation is important. The catches of paratenic hosts and the rate of infection vary with year. In addition, the kind of paratenic host fishes are different between the southern and northern areas of Japan. The paratenic hosts reported by patient are closely related to the catches of kinds of those fishes in the respective areas. Recently, cases by eating sardine are increasing in the southern area. Urticaria as a complication is related to the diagnostic rate, and intraperitoneal bacterial infection by the gastrointestinal perforation by worms is closely related to the prognosis.

Surface ultrastructure of larval Anisakidae (Nematoda: Ascaridoidea) and its identification by mensuration.

The surface ultrastructure of larval Anisakis type I, Anisakis type II, Raphidascaris, Contracaecum type A, Thynnascaris type A and Thynnascaris type B was examined by scanning electron microscopy. These species were identified clearly by the presence of a boring tooth, a mucron, and other morphological features. The means of the distances between transverse striations (DBTS) of larval Anisakis type I (5.45 +/- 0.125 micron), larval Raphidascaris (2.92 +/- 0.051 micron), and larval Contracaecum type A (1.68 +/- 0.056 micron) are significantly different (p less than 0.05). There was a correlation between the diameter of worm trunk (DOWT) and DBTS among these three larval types. In most cases a larva could be identified from the mean value of DBTS and DOWT even if obtained as a fragment from a patient.

Anti-parasite activity of nucleoside analogues: the metabolism of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani and its activity against amastigotes of Leishmania donovani in vitro.

Carbocyclic inosine is a potent inhibitor for the growth of the promastigote form of Leishmania tropica and Leishmania donovani. In culture, the EC50 values of carbocyclic inosine are 8.3 X 10(-8) and 1.3 X 10(-7) M for the promastigotes of L. tropica and L. donovani, respectively. On the other hand, it is less toxic towards mouse mammary tumor FM3A cells: the EC50 value is 2.7 X 10(-4)M. Carbocyclic inosine is metabolized by Leishmania promastigotes to give carbocyclic adenosine-5'-triphosphate(aristeromycin-5'-triphosphate) and carbocyclic guanosine-5'-triphosphate. This metabolic conversion provides a mechanism for the parasite-selective toxicity of carbocyclic inosine. Carbocyclic inosine was found to be active against L. donovani amastigotes in an in vivo-like cultivation in vitro.

[Ultrastructural changes of the tegument on Diphyllobothrium erinacei and Hymenolepis nana treated with paromomycin sulfate in vitro].

The effects of paromomycin sulfate on Diphyllobothrium erinacei and Hymenolepis nana in vitro were examined morphologically with a scanning and a transmission electron microscope. D. erinacei was incubated for 3 and 6 hours at 37 degrees C in a culture medium, 0.85% physiological saline solution, containing 0.5% paromomycin sulfate. H. nana was incubated in the same medium for 3 hours only. The concentration of paromomycin sulfate was set basing on the results which Kitamoto (1968) reported as the concentration level in feces after administrations of this drug in a clinical survey. The effect of the drug on the surface structure in both worms appeared markedly in the neck region. Mechanisms of breakdown on the tegument were supposed as follows. First, microtriches were disconnected from the tegumental surface and many vesicles were formed in the cytoplasm of the tegument. Finally, the tegument layer was excoriated to exposed the basal lamina. In 6 hours incubation, this surface of the worm suffered more damage than that in 3 hours. The damage of the basal lamina as in the case of D. latum expelled from a man by paromomycin (Y. Tongu et al.), however, could not be observed in the present study in vitro. It suggests that the destruction of basal lamina usually observed with the expelled worms from clinically treated human might be due to the combined effect of digestive enzymes secreted from host and the mechanical impact of intestinal peristalsis. Some of the vesicles in the tegument may originate from mitochondria because the fine structure of cristae were occasionally observed remaining in the vesicles.