ECG changes after rabbit coronavirus infection.

This study examines the electrocardiographic (ECG) changes following rabbit coronavirus (RbCV) infection. We have shown that infection with RbCV results in the development of myocarditis and congestive heart failure and that some survivors of RbCV infection go on to develop dilated cardiomyopathy in the chronic phase. Serial ECGs were recorded on 31 RbCV-infected rabbits. Measurements of heart rate; P-R interval; QRS duration; QTc interval; and P-, QRS-, and T-wave voltages were taken. The recordings were also examined for disturbances of conduction, rhythm, and repolarization. The acute and subacute phases were characterized by sinus tachycardia with depressed R- and T-wave voltages as well as disturbances of conduction, rhythm, and repolarization. In most animals in the chronic phase, the sinus rate returned to near-baseline values with resolution of the QRS voltage changes. The ECG changes observed during RbCV infection are similar to the spectrum of interval/segment abnormalities, rhythm disturbances, conduction defects, and myocardial pathology seen in human myocarditis, heart failure, and dilated cardiomyopathy. Because animals often died suddenly in the absence of severe clinical signs of congestive heart failure during the acute phase, RbCV infection may increase ventricular vulnerability, resulting in sudden cardiac death. RbCV infection may provide a rare opportunity to study sudden cardiac death in an animal model in which the ventricle is capable of supporting ventricular fibrillation, and invasive techniques monitoring cardiac function can be performed.

Control of glucan-induced systemic granulomatosis by cyclosporine A.

This study has shown that cyclosporine A (CyA), under certain conditions, is a powerful inhibitor of intravascular and extravascular monocyte/macrophage accumulation. Experiments were carried out in Lewis rats in which intravenous injection of particulate glucan calls forth a striking granulomatous response in lung, liver, and spleen and produces a marked stimulation of splenic erythro- and myelopoiesis. In agreement with the results of others, there was also a considerable elevation in monocyte/macrophage chemoattractant levels in the bronchoalveolar lavage fluid, which is held to be a key reaction in the pathogenesis of the histologic lesions. Treatment of the animals with subcutaneous injections of CyA prevented the rise in the chemoattractant activity and suppressed the granulomatous organ infiltration as well as the splenic hemopoiesis. The findings supply new insights into the activities of CyA and would support its clinical use in macrophage-dominated diseases.

An experimental model for myocarditis and congestive heart failure after rabbit coronavirus infection.

In a model for virus-induced myocarditis and congestive heart failure, rabbit coronavirus infection was divided into acute (days 2-5) and subacute (days 6-12) phases on the basis of day of death and pathologic findings. During the acute phase, the principal histologic lesions were degeneration and necrosis of myocytes, myocytolysis, interstitial edema, and hemorrhage. The severity of these changes increased in the subacute phase. Pleural effusion and congestion of the lungs and liver were also present at this time. Myocarditis was detected by day 9 and peaked by day 12. Heart weights and heart weight-to-body weight ratios were increased, and dilation of the right ventricular cavity became prominent early in infection and persisted. In contrast, dilation of the left ventricle occurred late in the subacute stage. Virus was isolated from infected hearts between days 2 and 12. These data suggest that rabbit coronavirus infection progresses to myocarditis and congestive heart failure.

Streptococcal cell wall-induced systemic disease. Beneficial effects of trans-bis(5-amidino-2-benzimidazolyl)ethene, a novel, macrophage-directed anti-inflammatory agent.

Previously bis(5-amidino-2-benzimidazolyl)methane (BABIM) was identified as a strong inhibitor of the multisystem inflammatory disease induced in Lewis rats by injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide (PG-APS). A BABIM derivative, trans-bis(5-amidino-2-benzimidazolyl)ethene (BBE), has attracted attention because of striking qualitative and quantitative differences in its activities when compared with the parent compound. BBE could control destructive tibial osteitis and necrotizing granulomatous splenitis and hepatitis, regardless if given in a preventive or curative mode. The compound had little effect on synovitis, however. BABIM, on the other hand, was active against synovitis and osteitis, but not against splenic granuloma formation. To be effective, it needed to be applied in a preventive mode. BBE caused a characteristic enlargement of PG-APS-laden splenic and hepatic macrophages suggesting that those cells represent targets of the inhibitor. BBE may be a powerful tool for the study of granulomatous lesions.

Novel pentamidine analogs in the treatment of experimental Pneumocystis carinii pneumonia.

We have recently demonstrated that substitution of imidazoline moieties for the amidine groups of pentamidine produces a molecule that is effective against rat Pneumocystis carinii pneumonia and that is apparently less toxic than pentamidine. For this reason, 10 novel imidazoline substituted compounds were evaluated for their effect against rat P. carinii pneumonia. While several of the new compounds were observed to have advantages over pentamidine in the treatment of disease in the rat model, only one compound stood out as a potential new clinical agent. Treatment for 2 weeks with intravenous (i.v.) doses of 1,3-di(4-imidazolino-2-methoxyphenoxy)propane (DIMP) at 1 mg/kg per day produced an anti-P. carinii pneumonia effect equivalent to i.v. doses of pentamidine at 10 mg/kg per day. Although pentamidine and one of the test drugs, 1,3-di(4-imidazolinophenoxy)propane, showed no activity against P. carinii pneumonia when administered per os, DIMP exhibited potent anti-P. carinii pneumonia activity when given by daily gavage doses of 40 and 25 mg/kg. DIMP retained significant activity when given every other day by a gavage dose of 25 mg/kg. No toxicity was observed with the drug at any of the dose levels or by either of the routes of administration. However, the low solubility of the drug prevented testing at higher i.v. doses. Our conclusion is that DIMP has the potential of providing a safer and more effective alternative to pentamidine for the treatment of P. carinii pneumonia.

Analogues of 1,5-bis(4-amidinophenoxy)pentane (pentamidine) in the treatment of experimental Pneumocystis carinii pneumonia.

A series of 33 analogues of the anti-Pneumocystis carinii drug 1,5-bis(4-amidinophenoxy)pentane (pentamidine) was synthesized for screening against a rat model of P. carinii pneumonia (PCP). Twenty-five of the compounds showed efficacy against PCP when compared to a saline-treated control group. Two compounds, 1,4-bis(4-amidinophenoxy)butane (butamidine, 6) and 1,3-bis(4-amidino-2-methoxyphenoxy)propane (DAMP, 16), were statistically more effective than the parent drug in treating PCP in the rat model of infection. In addition to their activity against PCP, the compounds were also evaluated for antitrypsin activity, ability to inhibit thymidylate synthetase, affinity for DNA, and toxicity. No correlation was observed between the tested molecular interactions of the diamidines and their effectiveness against PCP.

Sequential events in the pathogenesis of streptococcal cell wall-induced arthritis and their modulation by bis(5-amidino-2-benzimidazolyl)methane (BABIM).

This report builds on the authors' earlier discovery of bis(5-amidino-2-benzimidazolyl)methane (BABIM) as a strong suppressive agent for streptococcal cell wall fragment-induced arthritis in the Lewis rat. As a synthetic inhibitor of trypsinlike proteases, BABIM opens up a new route to the control of inflammatory joint disease. To gain a deeper insight into the function of the compound, the authors have now studied its influence on the sequential development of the joint changes and the associated lesions in spleen and liver. Bis(5-amidino-2-benzimidazolyl)methane is shown to block acute synovitis, to retard and reduce granuloma formation in spleen and liver, to decrease neutrophilic leukocytosis, and to diminish hemopoietic hyperplasia in the bone, and thus also to mitigate the distinctive osteoclastic and chondroclastic events. The compound does not interfere with the splenic immune response, the temporary rise in hepatocytic mitotic activity, or the organ deposition of streptococcal cell walls.

Treatment of experimental Pneumocystis carinii pneumonia with analogs of pentamidine.

Seven analogs of pentamidine were tested for their activity against an immunosuppressed rat model of Pneumocystis carinii pneumonia. Structural alterations of the pentamidine molecule included variations of the alkyl chain linking the two p-amidino phenoxy moieties and relocation of the amidine groups from the para to the meta position on the phenoxy rings. All analogs of pentamidine were active against P. carinii pneumonia when compared to a saline-treated control group. One derivative, 1, 4-di(4'-amidinophenoxy)butane, proved to be statistically more active than the parent drug.

Protease inhibitors suppress the in vitro and in vivo replication of rotavirus.

Rotaviruses are major causes of infectious gastroenteritis in humans and other animals. We found that a variety of protease inhibitors suppressed the replication of the SA-11 strain of rotavirus in MA-104 cell cultures. Three of these compounds, leupeptin, pentamidine, and bis (5-amidino-2-benzimidazolyl) methane (BABIM) also restricted the intestinal replication of the murine strain of rotavirus when protease inhibitor and virus were administered simultaneously to suckling mice. Repeated administration of BABIM resulted in significantly reduced levels of intestinal rotaviral antigen even if administration of the compound was begun as late as 48 h after viral inoculation. Additionally, BABIM-treated animals had significantly less intestinal replication of rotavirus than did placebo-treated controls when placed in a heavily rotavirus-contaminated environment. The use of protease inhibitors represents a novel approach to the control of this important gastrointestinal pathogen and is a potential modality for the prevention and treatment of diseases caused by other enteric viruses, for which proteolytic cleavage is necessary for efficient replication.

Suppression of streptococcal cell wall-induced arthritis by a potent protease inhibitor, bis(5-amidino-2-benzimidazolyl)methane.

Bis(5-amidino-2-benzimidazolyl)methane, a powerful synthetic trypsin inhibitor, proved to be highly effective in suppressing the arthritis induced by streptococcal cell wall fragments in Lewis rats. It reduced not only the degree of synovitis, osteitis, and hematopoietic hyperplasia in the distal extremities, but also the degree of associated granulomatous inflammation in the liver. The results suggest that trypsin-like proteases play an important role in this arthritis model and that inhibitors may be useful in the treatment of similar arthritic conditions in humans.

Amelioration of immune complex-mediated glomerulonephritis by synthetic protease inhibitors.

Proteases are involved in the pathogenesis of inflammatory diseases by participating in the activation of mediator systems and by producing proteolytic tissue injury. Homeostatic control of inflammation is accomplished in part by physiologic protease inhibitors. The authors investigated the effectiveness of a number of synthetic protease inhibitors in ameliorating the glomerular injury induced by immune complex-mediated glomerulonephritis in mice. Two amidine-type protease inhibitors, bis (5-amidino-2-benzimidazolyl)methane and 1,2-bis (5-amidino-2-benzimidazolyl)ethane, had the greatest effects. They caused a marked reduction in glomerular necrosis (P less than 0.001) but did not affect the amount or site of immune complex localization or leukocyte influx. The inhibition constants of the protease inhibitors against nine purified physiologic proteases were determined. These results were discussed in relation to the effectiveness of the protease inhibitors in reducing glomerular injury. This investigation indicates that the administration of synthetic protease inhibitors can have a beneficial effect on immune-mediated inflammatory injury.

Angiosarcoma of the uterus: a light microscopic, immunohistochemical, and ultrastructural study.

A 71-year-old woman who presented with severe vaginal bleeding was found to have a uterine angiosarcoma after total abdominal hysterectomy and bilateral salpingo-oophorectomy. The diagnosis was confirmed by immunohistochemical studies that showed positive staining of tumor cells for factor VIII-related antigen and by ultrastructural studies revealing vasoformative structures surrounded by a thin basal lamina. Primary vascular tumors of the uterus are rare. The existence of malignant vascular neoplasms of the uterus has been questioned, and at most nine previously reported cases have been generally accepted as valid examples of uterine angiosarcoma. Based on those cases, the prognosis of uterine angiosarcoma is poor. Our studies demonstrate that angiosarcoma does occur in the uterus. Our patient is presently alive with recurrent disease.

In vitro inhibition of human sarcoma cells' invasive ability by bis(5-amidino-2-benzimidazolyl)methane--a novel esteroprotease inhibitor.

Bis(5-amidino-2-benzimidazolyl)methane (BABIM) is a synthetic aromatic amidine compound which has a number of important biochemical effects, including inhibition of a family of esteroproteases (trypsin, urokinase, plasmin) previously linked to the complex process of tumor invasion. Previous work has suggested that exogenous natural protease inhibitors can block invasion of tumor cells across basement membranes (BM) in vitro. The authors studied the effect of BABIM on the human cell line HT-1080 with the use of a quantitative in vitro amnion invasion assay system. They have verified the ability of these cells to grow in nude mice and metastasize via the lymphatics or blood vessels on the basis of the route of administration of the inoculum. Cells which were able to actively cross the entire BM were trapped on filters and counted by both brightfield microscopy and by beta scintillation counting of cells whose DNA was labeled with tritiated thymidine. In agreement with either counting technique, BABIM, at a concentration of 10(-4) M, significantly inhibited invasion (P less than 0.005) over the 7-day course of the experiments. Under these conditions, the inhibitor was nontoxic and did not alter the attachment of the cells to the amniotic membrane. Furthermore, a highly significant inhibition of invasion (P less than 0.001) was also demonstrated across a variation in molar concentration of BABIM of more than 2 orders of magnitude. Most remarkably, cells were initially inhibited in their ability to invade in the presence of between 10(-9) and 10(-3) M BABIM. Measurement of Type IV specific collagenase in media from these cells shows a significant inhibition of activity in the presence of BABIM. These results suggest two, not necessarily exclusive, alternative interpretations: first, that inhibition of the proteolytic steps along the pathway of activation of basement membrane degrading enzymes results in inhibition of invasion; second, that arginine directed esteroproteases may work in concert with cellular collagenolytic metalloproteinases in the process of invasion by human tumor cells through native matrix barriers.

Suppression of respiratory syncytial virus infection in cotton rats by bis(5-amidino-2-benzimidazolyl)methane.

Intraperitoneal administration of bis(5-amidino-2-benzimidazolyl)methane at well-tolerated daily doses of 25 mg/kg subsequent to challenge and for 3 days thereafter effected over a 1-log reduction in the amount of virus recovered from lungs of cotton rats inoculated intratracheally with respiratory syncytial virus. When animals were immunosuppressed to prolong virus shedding, the reduction in recovered virus achieved with a 7-day dosing schedule of bis(5-amidino-2-benzimidazolyl)methane exceeded 2 logs.

Effect of synthetic protease inhibitors of the amidine type on cell injury by Rickettsia rickettsii.

To evaluate the importance of proteolytic activity in the pathogenesis of cell injury by Rickettsia rickettsii, a series of four aromatic amidine inhibitors of trypsin-like proteases were introduced into the plaque model. The compounds were shown to be active toward plaque reduction with their order of effectiveness parallel to their antitrypsin activity. One of the compounds, bis(5-amidino-2-benzimidazolyl)-methane, at a concentration of 10(-5) M demonstrated complete inhibition of plaque formation on day 6. Bis(5-amidino-2-benzimidazolyl)methane at the same concentration reduced cell injury even when added to the system after 72 h of rickettsial infection. The reduction in morbidity in guinea pigs experimentally infected with R. rickettsii and treated with bis(5-amidino-2-benzimidazolyl)methane as compared with morbidity in infected, untreated animals, comprised delay in the onset of fever and slightly fewer febrile animals. Because bis(5-amidino-2-benzimidazolyl)methane had no effect on phospholipase A2, the enzyme activity associated with penetration-induced cell injury, it is likely that a trypsin-like protease also plays an essential role either in the physiology of R. rickettsii or as its pathogenic mechanism.

Mucoepidermoid carcinoma in a cervical cyst: a case of branchiogenic carcinoma?

A patient with a primary oral epidermoid carcinoma with presumed neck metastasis is presented who at operation was found to have a cervical mucoepidermoid carcinoma arising in the wall of a benign cyst. The case for considering this tumor a primary branchioma of mucoepidermoid type is presented, and the criteria for making the diagnosis of branchiogenic carcinoma are discussed.

Enhancement of respiratory syncytial virus-induced cytopathology by trypsin, thrombin, and plasmin.

A series of proteases of diverse substrate specificity were tested for their effect on respiratory syncytial virus-induced cytopathology. Three of the enzymes, thrombin, plasmin, and trypsin, were able to augment significantly the fusion of virus-infected A549 cells. On a concentration basis, thrombin was the most active promoter, followed by plasmin and then trypsin. Hirudin, a specific thrombin inhibitor, blocked the fusion-enhancing property of thrombin, yet had no influence on the basal rate of fusion in the absence of the enzyme. By contrast, the amidine-type inhibitors of trypsin-like proteases, bis(5-amidino-2-benzimidazolyl)-methane (BABIM), blocked not only the thrombin effect, but also the fusion in the thrombin-free controls. The suppressive activity of BABIM was observed at concentrations so low as to exclude any direct inhibitory effect on thrombin itself. These results make it seem very likely that thrombin advances cell fusion by activating a BABIM-sensitive protease. Plasmin and trypsin can be expected to act in a similar manner.

Aromatic amidines: comparison of their ability to block respiratory syncytial virus induced cell fusion and to inhibit plasmin, urokinase, thrombin, and trypsin.

Two series of amidine derivatives consisting of a total of 24 compounds were examined for a correlation between their blocking effect on respiratory syncytial virus induced cell fusion and their inhibitory activity against selected trypsin-like protease. Although no correlation was evident between the two activities, several potentially important discoveries were made. A highly selective inhibitor of plasmin over thrombin (compound 10) was obtained, and a potent new blocker of virus-induced cell fusion (compound 22) was identified.