Enhanced platelet adherence and aggregation in Chagas' disease: a potential pathogenic mechanism for cardiomyopathy.

Spasm and thrombosis of the coronary microcirculation has been implicated in the pathogenesis of the cardiomyopathy of Chagas' disease. We demonstrate that increases in platelet adherence and aggregation accompany Trypanosoma cruzi infection and may contribute to the observed microvascular pathology. Scanning electron microscopy and radiolabeled platelets studies revealed that platelet adherence to T. cruzi-infected human endothelial cells was significantly increased when compared to controls (P = 0.024). In in vitro experiments, we determined the influence of infection on prostacyclin production, a marker of endothelial cell perturbation. The basal levels of 6-keto-prostaglandin F1 alpha was significantly greater in the supernatant of infected endothelial cells than in those of uninfected endothelial cells (P less than 0.05). The influence of infection was assessed on platelet aggregation at days 5 and 12 post-infection in A/J mice. Platelets from T. cruzi-infected mice were 2-6-fold more sensitive to aggregation induced by adenosine diphosphate and sodium arachidonate than controls. Thromboxane B2 levels in the plasma of infected mice were greater than controls. These data support the hypothesis that heightened platelet reactivity and endothelial cell dysfunction are associated with acute Chagas' disease and may cause coronary microvascular spasm and/or occlusion.

Trypanosoma cruzi: inhibition by spirogermanium hydrochloride.

Spirogermanium is an antineoplastic agent that has been shown to be useful for the treatment of a variety of solid tumors and Plasmodium falciparum infection. We found that this agent, at concentrations of 1-10 micrograms/ml, markedly inhibited the growth of epimastigotes of Trypanosoma cruzi. This inhibition of growth was seen in liver infusion tryptose cultures as well as on agar where colonial growth was inhibited markedly. Ultrastructural studies demonstrated that affected organisms were round and swollen and contained vacuoles, lamellar structures, and multivesicular bodies. Spirogermanium also significantly decreased the growth of intracellular amastigotes in myotubes. Pretreatment of myotubes with the agent protected them from infection with trypomastigotes but tachyzoites of Toxoplasma sp. readily infected pretreated cells. These data suggest that spirogermanium may be useful as a chemotherapeutic agent against T. cruzi.

Studies of the etiology of Crohn's disease using athymic nude mice.

Following injection of Crohn's disease tissue filtrates, lymphomas and hyperplastic lymph nodes developed in 16% of athymic nude (nu/nu) mice; whereas only 4% of control nude mice developed lymphadenopathy (p less than 0.025). One hundred forty coded sera from 111 patients (Crohn's disease = 36, ulcerative colitis = 28, diarrheal and other controls = 47) were assayed by indirect immunofluorescence for immunoreactivity with the lymphomas and hyperplastic lymph nodes. Coded sections were examined by two observers and scored on a 0 to 3 + scale. Fifty-four percent of the sera from patients with Crohn's disease were reactive with the Crohn's disease induced lymphoma by this assay. Eighty percent of sera from patients with symptomatic Crohn's disease were positive, whereas 22% of sera from patients in remission were positive. Sixty-six percent of sera from patients with symptomatic Crohn's disease reacted against hyperplastic lymph nodes induced by Crohn's disease filtrates. In contrast, only one control serum (from a patient with ulcerative colitis) reacted with the lymphomas or hyperplastic lymph nodes. Lymphomas induced by other means or arising spontaneously did not show immunofluorescence with Crohn's disease or control sera. Electron microscopy revealed C-type viral particles in five lymphomas induced by Crohn's disease filtrates and in one control lymphoma, but not in five hyperplastic lymph nodes and five control lymph nodes. Absorption of Crohn's disease sera with control lymphoma or with murine leukemia virus infected fibroblasts did not diminish immunoreactivity, whereas similar absorption with lymphomas induced by Crohn's disease filtrates abolished the immunofluorescence. These studies indicate that Crohn's disease tissue, when injected into athymic nude mice, induces lymphoid hyperplasia and lymphomas that contain an antigen(s) recognized by Crohn's disease sera.

The effect of adenosine analogues on the in vitro growth of Trypanosoma cruzi.

Trypanosoma cruzi (Brazil strain) was maintained in liver infusion tryptose and medium supplemented with 5 or 10% foetal calf serum at 27 degrees C on a rotating shaker platform. 85 to 95% of the organisms under these conditions are epimastigotes and the medium supported logarithmic growth for up to 24 hours. The effect of S-isobutyl adenosine and Sinefungin against cultured T. cruzi epimastigotes was studied: growth rate was slowed by both in a dose-dependent fashion; 500 micrometer Sinefungin caused complete inhibition which was irreversible after 24-hour exposure but the effect of S-isobutyl adenosine (100 micrometer) was reversible. Motility and morphology appeared to be unaffected.

Taxol, a microtubule stabilizing agent, blocks the replication of Trypanosoma cruzi.

Taxol, an experimental antitumor agent and stabilizer of microtubules, inhibits in vitro replication of the human pathogenic hemoflagellate Trypanosoma cruzi. Micromolar concentrations of the drug prevent the completion of cell division in these organisms but allow the multiplication of cell organelles such as the nucleus, kinetoplast, and flagellum. The result is the formation of motile organisms that have extra organelles but cannot fully replicate. Division proceeds to a relatively fixed locus on the long axis of the organism, suggesting the presence of a specific affected structure or function at this site. It is postulated that taxol produces these effects by stabilizing a portion of the microtubular cytoskeleton of T. cruzi.

Transcription of adenovirus RNA in permissive and nonpermissive infections.

The mechanism of blocked replication of human adenoviruses in monkey cells was examined. Previous experiments have placed the replicative block at the level of transcription of translation of adenovirus mRNA. Coinfection of the monkey cells with simian virus 40 enhances adenovirus replication in these cells. We compared the adenovirus mRNA transcribed during infection of permissive human cells and enhanced and unenhanced monkey cells. Adenovirus mRNA from enhanced monkey cells appeared to be identical to adenovirus mRNA from human cells. This indicated that simian virus 40 coinfection did not overcome the blocked replication by substituting for a missing adenovirus transcript. Comparison of adenovirus mRNA from enhanced and unenhanced monkey cell infection revealed two types of transcriptional discrepancies. There was a decrease in both the complexity and the relative abundance of several regions of the enhanced adenovirus mRNA. However, neigher of these transcriptional defects was sufficient to totally explain the difference in yield of infectious virus and viral protein seen in these two types of infection.

Infective endocarditis caused by Streptococcus mutans. A complication of idiopathic hypertrophic subaortic stenosis.

Three patients with endocarditis caused by Streptococcus mutans were seen during a six-month period. All had clinical features of subacute bacterial endocarditis, including fever, heart murmurs, and positive blood cultures. One had underlying aortic insufficiency and two had idiopathic hypertrophic subaortic stenosis. All patients were treated with parenteral antibiotics and were cured. Streptococcus mutans is a pleomorphic, microaerophilic organism that is associated with dental caries and plaque. Differentiation of S mutans from enterococcal endocarditis is important because the former condition can be treated for a shorter period of time with penicillin alone, without the addition of aminoglycoside antibiotics.

Persistent adenovirus infections of nonpermissive monkey cells.

Persisten infections of monkey cells have been established by using two serotypes of human adenovirus. The persistently infected cells show no morpho logical changes, but continue ot produce low titers of infectious adenovirus. The inapparent infection can, at any time, be converted to a cytolytic productive one by superinfection with simian virus 40. Persistence in this system does not appear to result from multiple rounds of lytic infection, nor is it mediated by production of defective interfering particles. The persistently infected cells do not possess the characteristics of oncogenic transformation. Results of these studies also whow that the nonpermissiveness of monkey cells to adenovirus replication can be partially overcome by infection at high multiplicity.

Regulation of in vitro insulin release from a transplantable Syrian hamster insulinoma.

An insulin-producing islet cell tumor of the Syrian hamster has been studied in vitro for its capacity to respond to known stimuli of insulin release. Insulin secretion during short term incubation and perifusion of fragments of tumor was detected by radioimmunoassay. Insulin release was increased 2-4 fold by 40 mM potassium in the presence of calcium, glucose (22 mM), glucagon (0.3-3.0 muM), N6,02'-dibutyryl adenosine 3',5'-monophosphate (cAMP; 6mM), and theophylline (10 mM). Concentrations of glucagon that induced insulin release were also effective in activating adenylate cyclase in the membranes of tumor cells. Thus, this tumor appears to possess a cAMP-mediated mechanism for insulin release. Somatostatin (0.8-25 mum) inhibited glucagon-induced insulin release without altering basal or glucagon stimulated adenylate cyclase activity. It would appear that inhibition of glucagon induced insulin release by somatostatin is not mediated by adenylate cyclase. We propose that insulin release by this tumor is sufficiently similar to that found in normal islets so as to make it a suitable model for biochemical studies that require large quantities of homogeneous tissue.

Retention of endocrine function by an insulin-secreting pancreatic islet cell tumour from Syrain hamster through serial transplantation in nude mice.

An insulin-secreting islet cell tumour of the Syrian hamster has been transplanted serially in the congenitally immune-deficient nude mouse, in order to test the potential usefulness of this mouse mutant as a graft carrier of heterologous tumours with stable differentiated phenotypes. The incidence of tumour growth was very high, and the hamster tumour retained its functional and histologic characteristics during consecutive passages in nude mice. These results show that nude mice may be useful carriers of differentiated tumours from non-inbred species including man, and for the isolation of cell lines from such tumours.

Synthesis of type 2 adenovirus DNA in the presence of cycloheximide.

Adenovirus type 2 DNA synthesis, either in permissive human cells or nonpermissive monkey cells, becomes independent of protein synthesis after the appearance of progeny viral DNA. In the presence of cycloheximide, semiconservative replication and initiation of progeny molecules can occur.

Studies of the mechanism of enhancement of human adenovirus infection in monkey cells by simian virus 40.

The defect which prevents human adenovirus replication in monkey cells and the mechanism whereby this restriction is overcome by coinfection with simian virus 40 (SV40) have been studied. Adenovirus capsid proteins are not synthesized efficiently in monkey cells in the absence of SV40. Adenovirus "enhancement" by SV40 was found to be the product of increased efficiency of replication by a small percentage of cells. This enhancement effect apparently occurred only when SV40 and adenovirus infected the same cells. These findings suggest that the replicative block occurs prior to virion assembly, and an accompanying report seeks to locate the site of restriction more precisely at the post-transcriptional level.

Synthesis of viral ribonucleic acid during restricted adenovirus infection.

The mechanism by which simian virus 40 converts the abortive adenovirus type 7 infection of monkey cells into an efficient lytic infection has been investigated. Analysis of ribonucleic acid (RNA) synthesis during unenhanced and enhanced infection of monkey cells has shown that adenovirus RNA synthesized in the abortive infection contains both "early" and "late" sequences. In hybridization competition experiments, early adenovirus RNA from human cells prevented the hybridization of only 20% of the adenovirus RNA transcribed in unenhanced infection. Further, the RNA from unenhanced cells was able to completely block the hybridization of RNA synthesized during enhanced infection. Finally, virus-associated RNA, which is a late RNA transcribed in lytic adenovirus infection, is also produced in the unenhanced infection. An accompanying paper describes a marked deficiency in adenoviral capsid protein synthesis in the unenhanced infection. We conclude that RNA sequences, which are sufficient to code for the synthesis and assembly of structural proteins of adenovirus, are transcribed but are not efficiently translated in the unenhanced adenovirus infection of monkey cells.