Risk of Venous Thromboembolism Following Hemorrhagic Fever With Renal Syndrome: A Self-controlled Case Series Study.

Background: Bleeding is associated with viral hemorrhagic fevers; however, thromboembolic complications have received less attention. Hemorrhagic fever with renal syndrome (HFRS) is a mild viral hemorrhagic fever caused by Puumala hantavirus. We previously identified HFRS as a risk factor for myocardial infarction and stroke, but the risk for venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is unknown. Methods: Personal identity numbers from the Swedish HFRS database were cross-linked with the National Patient register to obtain information on all causes for hospitalization during 1964 to 2013. The self-controlled case series method was used to calculate the incidence rate ratio (IRR) for first VTE, DVT, and PE during 1998 to 2013. Results: From 7244 HFRS patients, there were 146 with a first VTE of which 74 were DVT and 78 were PE, and 6 patients had both DVT and PE. The overall risk for a VTE was significantly higher during the first 2 weeks following HFRS onset, with an IRR of 64.3 (95% confidence interval [CI], 36.3-114). The corresponding risk for a DVT was 45.9 (95% CI, 18-117.1) and for PE, 76.8 (95% CI, 37.1-159). Sex interacted significantly with the association between HFRS and VTE, with females having a higher risk compared with males. Conclusions: A significantly increased risk for VTE was found in the time period following HFRS onset. It is important to keep this in mind and monitor HFRS patients, and possibly other viral hemorrhagic fever patients, for early symptoms of VTE.

Increased Thrombopoiesis and Platelet Activation in Hantavirus-Infected Patients.

BACKGROUND: Thrombocytopenia is a common finding during viral hemorrhagic fever, which includes hemorrhagic fever with renal syndrome (HFRS). The 2 main causes for thrombocytopenia are impaired thrombopoiesis and/or increased peripheral destruction of platelets. In addition, there is an increased intravascular coagulation risk during HFRS, which could be due to platelet activation. METHODS: Thrombopoiesis was determined by quantification of platelet counts, thrombopoietin, immature platelet fraction, and mean platelet volume during HFRS. The in vivo platelet activation was determined by quantification of soluble P-selectin (sP-selectin) and glycoprotein VI (sGPVI). The function of circulating platelets was determined by ex vivo stimulation followed by flow cytometry analysis of platelet surface-bound fibrinogen and P-selectin exposure. Intravascular coagulation during disease was determined by scoring for disseminated intravascular coagulation (DIC) and recording thromboembolic complications. RESULTS: The levels of thrombopoietin, immature platelet fraction, and mean platelet volume all indicate increased thrombopoiesis during HFRS. Circulating platelets had reduced ex vivo function during disease compared to follow-up. Most interestingly, we observed significantly increased in vivo platelet activation in HFRS patients with intravascular coagulation (DIC and thromboembolic complications) as shown by sP-selectin and sGPVI levels. CONCLUSIONS: HFRS patients have increased thrombopoiesis and platelet activation, which contributes to intravascular coagulation.

Increased risk of acute myocardial infarction and stroke during hemorrhagic fever with renal syndrome: a self-controlled case series study.

BACKGROUND: We recently observed that cardiovascular causes of death are common in patients with hemorrhagic fever with renal syndrome (HFRS), which is caused by hantaviruses. However, it is not known whether HFRS is a risk factor for the acute cardiovascular events of acute myocardial infarction (AMI) and stroke. METHODS AND RESULTS: Personal identification numbers from the Swedish HFRS patient database (1997-2012; n=6643) were cross-linked with the National Patient Register from 1987 to 2011. Using the self-controlled case series method, we calculated the incidence rate ratio of AMI/stroke in the 21 days after HFRS against 2 different control periods either excluding (analysis 1) or including (analysis 2) fatal AMI/stroke events. The incidence rate ratios for analyses 1 and 2 for all AMI events were 5.53 (95% confidence interval [CI], 2.6-11.8) and 6.02 (95% CI, 2.95-12.3) and for first AMI events were 3.53 (95% CI, 1.25-9.96) and 4.64 (95% CI, 1.83-11.77). The incidence rate ratios for analyses 1 and 2 for all stroke events were 12.93 (95% CI, 5.62-29.74) and 15.16 (95% CI, 7.21-31.87) and for first stroke events were 14.54 (95% CI, 5.87-36.04) and 17.09 (95% CI, 7.49-38.96). The majority of stroke events occurred in the first week after HFRS. Seasonal effects were not observed, and apart from 1 study, neither sex nor age interacted with the associations observed in this study. CONCLUSIONS: There is a significantly increased risk for AMI and stroke in the immediate time period after HFRS. Therefore, HFRS patients should be carefully monitored during the acute phase of disease to ensure early recognition of symptoms of impending AMI or stroke.

Puumala virus infections associated with cardiovascular causes of death.

We studied the causes of death of patients in Sweden with diagnoses of hemorrhagic fever with renal syndrome (HFRS) during 1997-2009. Cardiovascular disorders were a common cause of death during acute-phase HFRS and were the cause of death for >50% of those who died during the first year after HFRS.

Are therapeutic human mesenchymal stromal cells compatible with human blood?

Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture-expanded human MSCs elicit an innate immune attack, termed instant blood-mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell-passage number. Short-term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D-dimer or acute-phase reactants with the currently applied dose of 1.0-3.0 × 10(6) cells per kilogram. Culture-expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose-dependent and increases after prolonged ex vivo expansion. Currently applied doses of low-passage clinical-grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells.

Crimean-Congo hemorrhagic fever virus activates endothelial cells.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes viral hemorrhagic fever with high case-fatality rates and is geographically widely distributed. Due to the requirement for a biosafety level 4 (BSL-4) laboratory and the lack of an animal model, knowledge of the viral pathogenesis is limited. Crimean-Congo hemorrhagic fever (CCHF) is characterized by hemorrhage and vascular permeability, indicating the involvement of endothelial cells (ECs). The interplay between ECs and CCHFV is therefore important for understanding the pathogenesis of CCHF. In a previous study, we found that CCHFV-infected monocyte-derived dendritic cells (moDCs) activated ECs; however, the direct effect of CCHFV on ECs was not investigated. Here, we report that ECs are activated upon infection, as demonstrated by upregulation of mRNA levels for E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Protein levels and cell surface expression of ICAM1 responded in a dose-dependent manner to increasing CCHFV titers with concomitant increase in leukocyte adhesion. Furthermore, we examined vascular endothelial (VE) cadherin in CCHFV-infected ECs by different approaches. Infected ECs released higher levels of interleukin 6 (IL-6) and IL-8; however, stimulation of resting ECs with supernatants derived from infected ECs did not result in increased ICAM1 expression. Interestingly, the moDC-mediated activation of ECs was abrogated by addition of neutralizing tumor necrosis factor alpha (TNF-α) antibody to moDC supernatants, thereby identifying this soluble mediator as the key cytokine causing EC activation. We conclude that CCHFV can exert both direct and indirect effects on ECs.

Endothelial activation and repair during hantavirus infection: association with disease outcome.

BACKGROUND: Endothelial activation and dysfunction play a central role in the pathogenesis of sepsis and viral hemorrhagic fevers. Hantaviral disease is a viral hemorrhagic fever and is characterized by capillary dysfunction, although the underlying mechanisms for hantaviral disease are not fully elucidated. METHODS: The temporal course of endothelial activation and repair were analyzed during Puumala hantavirus infection and associated with disease outcome and a marker for hypoxia, insulin-like growth factor binding protein 1 (IGFBP-1). The following endothelial activation markers were studied: endothelial glycocalyx degradation (syndecan-1) and leukocyte adhesion molecules (soluble vascular cellular adhesion molecule 1, intercellular adhesion molecule 1, and endothelial selectin). Cytokines associated with vascular repair were also analyzed (vascular endothelial growth factor, erythropoietin, angiopoietin, and stromal cell-derived factor 1). RESULTS: Most of the markers we studied were highest during the earliest phase of hantaviral disease and associated with clinical and laboratory surrogate markers for disease outcome. In particular, the marker for glycocalyx degradation, syndecan-1, was significantly associated with levels of thrombocytes, albumin, IGFBP-1, decreased blood pressure, and disease severity. CONCLUSIONS: Hantaviral disease outcome was associated with endothelial dysfunction. Consequently, the endothelium warrants further investigation when designing future medical interventions.

Crimean Congo hemorrhagic fever virus infects human monocyte-derived dendritic cells.

For some patients infection with Crimean Congo hemorrhagic fever virus (CCHFV) causes a severe disease characterized by fever, vascular leakage and coagulopathy. Knowledge of CCHF pathogenesis is limited and today there is no information about the specific target cells of CCHFV. In this study we analyzed the permissiveness of human peripheral blood mononuclear cells (PBMCs) including monocyte-derived dendritic cells (moDCs) to CCHFV infection. Interestingly, we found that moDCs are the most permissive to CCHFV infection and this infection induced cytokine release from moDCs. Furthermore, supernatants from infected moDCs were found to activate human endothelial cells.

Basolateral entry and release of Crimean-Congo hemorrhagic fever virus in polarized MDCK-1 cells.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an etiological agent of a disease with mortality rates in patients averaging 30%. The disease is characterized by fever, myalgia, and hemorrhage. Mechanisms underlying the hemorrhage have to our knowledge not been elucidated for CCHFV. Possibly, a direct or indirect viral effect on tight junctions (TJ) could cause the hemorrhage observed in patients, as TJ play a crucial role in vascular homeostasis and can cause leakage upon deregulation. Moreover, there is no knowledge regarding the site of entry and release of CCHFV in polarized epithelial cells. Such cells represent a barrier to virus dissemination within the host, and as a site of viral entry and release, they could play a key role in further spread. For the first time, we have shown preferential basolateral entry of CCHFV in Madin-Darby canine kidney 1 (MDCK-1) epithelial cells. Furthermore, we demonstrated basolateral release of CCHFV in polarized epithelial cells. Interestingly, by measuring transepithelial electrical resistance, we found no effect of CCHFV replication on the function of TJ in this study. Neither did we observe any difference in the localization of the TJ proteins ZO-1 and occludin in CCHFV-infected cells compared to mock-infected cells.