Thomas M. Cosgriff

Hemorrhagic fever virus-induced changes in hemostasis and vascular biology.

Viral hemorrhagic fever (VHF) denotes a virus-induced acute febrile, hemorrhagic disease reported from wide areas of the world. Hemorrhagic fever (HF) viruses are encapsulated, single-stranded RNA viruses that are associated with insect or rodent vectors whose interaction with humans defines the mode of disease transmission. There are 14 HF viruses, which belong to four viral families: Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae. This review presents, in order, the following aspects of VHF: (1) epidemiology, (2) anomalies of platelets and coagulation factors, (3) vasculopathy, (4) animal models of VHFs, (5) pathogenic mechanisms, and (6) treatment and future studies. HF viruses produce the manifestations of VHFs either by direct effects on cellular functions or by activation of immune and inflammatory pathways. In Lassa fever, Rift Valley fever and Crimean–Congo HF, the main feature of fatal illness appears to be impaired/delayed cellular immunity, which leads to unchecked viremia. However, in HF with renal syndrome and dengue HF, the immune response plays an active role in disease pathogenesis. The interplay of hemostasis, immune response, and inflammation is very complex. Molecular biologic techniques and the use of animal models have helped to unravel some of these interactions.

Pathogenesis of Rift Valley fever in rhesus monkeys : role of interferon response

SummaryRhesus monkeys inoculated intravenously with Rift Valley fever (RVF) virus presented clinical disease syndromes similar to human cases of RVF. All 17 infected monkeys had high-titered viremias but disease ranged from clinically inapparent to death. Three (18%) RVF virus-infected monkeys developed signs of hemorrhagic fever characterized by epistaxis, petechial to purpuric cutaneous lesions, anorexia, and vomiting prior to death. The 14 remaining monkeys survived RVF viral infection but, 7 showed clinical signs of illness characterized by diminished food intake, cutaneous petechiae, and occasional vomiting. The other 7 monkeys showed no evidence of clinical disease. All monkeys had detectable serum interferon 24–30 h after infection, but 4 of 7 monkeys that did not develop clinical illness had serum interferon titers within 12h after infection. In lethally infected macaques, indices of hepatic function and blood coagulation were abnormal within 2 days, implicating early pathogenetic events as critical determinants of survival. Serum transferase values were elevated in proportion to severity of clinical disease and outcome of infection. Both myocardial damage and laboratory evidence consistent with disseminated intravascular coagulation were present in fatal infections. All surviving monkeys developed neutralizing antibodies to RVF virus 4–7 days after infection, and this coincided with termination of viremia. Two fatally infected monkeys were viremic until death on days 6 and 8, and the third cleared viremia on day 5 and developed antibody on day 6 but died on day 15. There was a significant correlation between a delayed interferon response and mortality, suggesting that the early appearance of interferon was influential in limiting the severity of disease.

Experience with intravenous ribavirin in the treatment of hemorrhagic fever with renal syndrome in Korea

Abstract Results of a clinical study using intravenous (IV) ribavirin for treating Department of Defense personnel with hemorrhagic fever with renal syndrome (HFRS) acquired in Korea from 1987 to 2005 were reviewed to determine the clinical course of HFRS treated with IV ribavirin. A total of 38 individuals enrolled in the study had subsequent serological confirmation of HFRS. Four of the 38 individuals received three or fewer doses of ribavirin and were excluded from treatment analysis. Of the remaining 34 individuals, oliguria was present in one individual at treatment initiation; none of the remaining 33 subjects developed oliguria or required dialysis. The mean peak serum creatinine was 3.46mg/dl and occurred on day 2 of ribavirin therapy. Both the peak serum creatinine and the onset of polyuria occurred on mean day 6.8 of illness. Reversible hemolytic anemia was the main adverse event of ribavirin, with a ≥25% decrease in hematocrit observed in 26/34 (76.5%) individuals. While inability to adjust for all baseline variables prevents comparison to historical cohorts in Korea where oliguria has been reported in 39–69% cases and dialysis required in approximately 40% HFRS cases caused by Hantaan virus, the occurrence of 3% oliguria and 0% dialysis requirement in the treatment cohort is supportive of a previous placebo-controlled HFRS trial in China where IV ribavirin given early resulted in decreased occurrence of oliguria and decreased severity of renal insufficiency.

Platelet dysfunction contributes to the haemostatic defect in haemorrhagic fever with renal syndrome

To characterize further the nature of haemostatic impairment in haemorrhagic fever with renal syndrome, we assessed platelet function in 9 patients in whom the diagnosis was serologically confirmed. Defective platelet aggregation was demonstrated in every patient. An abnormality of the granule release reaction was demonstrated in all of 7 patients tested. Gel-filtered platelets from a normal subject showed normal aggregation in plasma from a patient with impaired aggregation, which is evidence for an intrinsic platelet defect, and against the presence of a circulating inhibitor in this patient.

The hemostatic derangement produced by T-2 toxin in guinea pigs☆☆☆

T-2 toxin produced significant coagulation abnormalities when administered parenterally to Hartley strain guinea pigs. The animals developed depressed activity of all coagulation factors except fibrinogen. Platelet aggregation in whole blood was depressed in response to ADP and collagen. The animals also exhibited an initial rise followed by a fall in hematocrit level, leukocytosis, and a decrease in platelet count. These changes were detectable within hours of toxin administration, reached a maximum at 24 hr, and returned to normal over the next 2 days. Pretreatment of animals with vitamin K1 had no effect on the activity of coagulation factors. The activated partial thromboplastin time of dilutions of plasma from animals given T-2 toxin with plasma from control animals revealed a pattern which pointed to a deficiency of coagulation factors as the principal cause of prolonged clotting times in treated animals. The presence of a weak circulating anticoagulant could not be ruled out. The addition of T-2 to plasma and blood of normal animals in a concentration of 1 microgram/ml had no effect on clotting times or platelet aggregation.

Immune serum increases arenavirus replication in monocytes

The U937 monocytic cell line was used to determine whether antibodies could facilitate infection and replication of the arenaviruses, Pichinde virus (PV) and Lassa fever virus (LFV). When high dilutions of PV-immune serum were added to cultures simultaneously with PV inoculum, virus replication was dramatically (1000-fold) increased. Low dilutions of this antiserum neutralized the virus. LFV also replicated in U937 cells. The presence of LFV-specific immune serum in the growth medium increased the viral titre as much as 10,000-fold. Addition of heat-aggregated IgG partially inhibited antibody-mediated enhancement, probably by inhibiting the binding of immune complexes to the monocytic cells.

Changes in populations of immune effector cells during the course of haemorrhagic fever with renal syndrome

To characterize the immune response in haemorrhagic fever with renal syndrome, serial changes in immune effector cells were measured in 14 patients. Significant findings included initial elevations of all major leucocyte populations, increases in suppressor T cells and B cells, decreases in helper/suppressor cell ratios, and a dramatic increase in activated T cells. These changes were most marked in severely ill patients. Changes reverted to normal over approximately one week.

The hemostatic derangement produced by T-2 toxin in cynomolgus monkeys.

T-2 toxin, a mycotoxin produced by several strains of the genus Fusarium, has been implicated as a cause of serious illness in both animals and man. Hemorrhage is a feature of the syndromes which have been described. An LD20 dose of T-2 was administered im to adult cynomolgus monkeys. This resulted in prolongation of prothrombin and activated partial thromboplastin times and a decrease in multiple coagulation factors. These changes were detected within hours of toxin administration, were maximal at 24 hr, and returned to normal over the next 3 days. Fibrin-fibrinogen degradation products were not detected at any time point. Repeated phlebotomy produced a significantly greater increase in platelet count in control monkeys, which could be taken as evidence for an effect of toxin on platelet kinetics. In treated animals, the hematocrit level declined by about 10%, but a similar decrease occurred in control animals. The white blood cell count increased 4 to 5 times over pretreatment values. Despite the changes in multiple laboratory parameters, treated monkeys did not exhibit clinical evidence of hemorrhage. In three animals which died as a result of toxicosis, necropsy revealed mild petechial hemorrhage involving the colon and heart, as well as necrosis of lymphoid tissues.

The Antithrombin III Content of Cryoprecipitate Prepared from Blood Collected with and without Heparin

Abstract. Antithrombin III (AT III) is a plasma protein which acts as the principal inhibitor of thrombin and is a major modulator of intravascular coagulation. Hereditary deficiency of AT III leads to recurrent episodes of thromboembolism. Acquired deficiency of AT III occurs in persons with a variety of conditions, including severe liver disease and disseminated intravascular coagulation. Replacement of AT III may be important in some deficient persons. To determine if cryoprecipitate is a useful source of AT III, we measured the AT III content of cryoprecipitate prepared from citrate phosphate dextrose blood using coagulation and fluorogenic assays and immunoassays. Using the fluorogenic assay, we also determined the effect of adding heparin to blood on the cryoprecipitation of AT III. Functional and antigenic AT III levels were similar to those of normal plasma in all citrate phosphate dextrose blood units tested, indicating that AT III is not concentrated in cryoprecipitate. Heparin had no effect on the cryoprecipitation of AT III.

Rhesus differential susceptibility to endotoxin is not associated with activation of plasma prekallikrein.

This study is part of a project aimed at understanding individual responses to acute endotoxemia in a catheter-free rhesus (Macaca mulatta) model of inflammation. In the previous study [J. Endotoxin Res. 2 (1995) 411-420.], we showed that of 14 endotoxin 0111:B4 (ETX)-infused monkeys, only three died at < 13.5 h and one at 6 days postinfusion. Doses of ETX correlated neither with the magnitude of hypotension nor with rhesus outcome. Survival (and death at 6 days) or death at < 13.5 h was rather associated with controllable or uncontrollable rise of plasma levels of proinflammatory cytokines and reversible or irreversible shock. In the current study, we used plasmas of 5 survivors and of one of the monkeys that died at < 13.5 h (each infused with 3 X 10(6) EU ETX/kg), and of two saline control monkeys of the previous study. We analyzed changes in parameters of coagulation and contact systems. After ETX infusion, activated partial thromboplastin time (APTT) and prothrombin time (PT) values increased modestly in survivors but markedly in the nonsurvivor; responses of platelet counts and levels of fibrinogen, antithrombin, alpha2-macroglobulin (alpha2M), Cl-inhibitor (C1INH) and alpha1 -antitrypsin were similar in survivors and the nonsurvivor; the rate of plasma prekallikrein (PK) activation measured by hydrolysis of the kallikrein (KAL) substrate D-Pro-Phe-Arg-p-nitroanilide was not altered by ETX infusion; and the distribution of PK activation products, analyzed by MAb 13G11/immunoblotting in plasmas with or without artificial activation, was similar in survivors and the nonsurvivor. Responses in controls were relatively stable. Since we used defined experimental conditions, this primate model has the potential to be useful to study further correlation of inflammatory parameters with differential outcome.