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Pathophysiology of a severe case of Puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant
Antiviral Research 111 (2014) 23–25
Contents lists available at ScienceDirect
Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
Short Communication
Pathophysiology of a severe case of Puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant Antti Vaheri a,b,⇑, Tomas Strandin a, Anne J. Jääskeläinen a,b, Olli Vapalahti a,b,c, Hanna Jarva a,d, Marja-Liisa Lokki a, Jaakko Antonen e, Ilona Leppänen e, Satu Mäkelä e,f, Seppo Meri a,d, Jukka Mustonen e,f a
Haartman Institute, University of Helsinki, Finland Department of Virology and Immunology, Helsinki University Central Hospital Laboratory, Finland Department of Veterinary Biosciences, University of Helsinki, Finland d Immunobiology Research Program, University of Helsinki, Finland e Tampere University Hospital, Finland f School of Medicine, University of Tampere, Finland b c
a r t i c l e
i n f o
Article history: Received 30 May 2014 Revised 11 August 2014 Accepted 14 August 2014 Available online 3 September 2014 Keywords: Hantavirus Pathophysiology Capillary leakage Complement activation Bradykinin Icatibant
a b s t r a c t We recently described a patient with very severe Puumala hantavirus infection manifested by capillary leakage syndrome and shock. He was successfully treated with the bradykinin receptor antagonist, icatibant (Antonen et al., 2013). Here we report analysis of the pathophysiology which indicated pronounced complement activation, prolonged leukocytosis, extensive fibrinolysis, circulating histones, and defects in liver function. The patient had an uncommon HLA-phenotype, which may have contributed to the severe course of the disease. Ó 2014 Elsevier B.V. All rights reserved.
Introduction The patient was a previously healthy 37-year-old Finnish male, except he had undergone 8 years earlier splenectomy due to congenital spherocytosis. He was admitted to hospital on March 10th 2011 due to acute Puumala virus (PUUV) infection. He suffered from a severe capillary leakage, shock, and multiorgan failure including kidney, liver and respiratory failure (Antonen et al., 2013). He was treated in the intensive care unit (ICU) until 23rd March 2011 and the total hospital care lasted until April 4th 2011. The treatment in the ICU included mechanical ventilation, continuous renal replacement therapy, massive fluid replacement, vasoactive drugs, inhaled nitric oxide, corticosteroids and ceftriaxone. He also got one subcutaneous dose of icatibant. The rationale was that a central pathophysiological aberration in hantavirus ⇑ Corresponding author at: Department of Virology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland. Tel.: +358 50 448 2850; fax: +358 2941 26491. E-mail address: antti.vaheri@helsinki.fi (A. Vaheri). http://dx.doi.org/10.1016/j.antiviral.2014.08.007 0166-3542/Ó 2014 Elsevier B.V. All rights reserved.
infections is increased capillary permeability. Bradykinin promotes permeability in many pathological conditions. Icatibant is a selective antagonist of the bradykinin type 2 receptor, which is indicated for the treatment of acute episodes of hereditary angioedema. During the next 24 h after dosing of icatibant the condition of the patient stabilized, followed by gradual improvement and a full recovery (Antonen et al., 2013).
2. Virological parameters PUUV is the only hantaviral pathogen in Finland and is carried by bank voles (Mustonen et al., 2013). The data in Table 1 confirm that the patient had acute PUUV infection: IgM antibodies, typical granular fluorescence pattern in immunofluorescence antibody test (IFAT) and high IgG-antibody avidity ratio (Vaheri et al., 2008). No PUUV RNA was detected in the serum. The patient had no antibodies to the following zoonosis-related viruses: cowpox, Ljungan, lymphocytic choriomeningitis or tick-borne encephalitis virus and thus no coinfections with these viruses were detected to explain the unusually severe disease.
24
A. Vaheri et al. / Antiviral Research 111 (2014) 23–25
Table 1 Biomarkers: virology and complement. Date of sample Test
Unit
PUUV IgG IFA titer PUUV IgG IFA patterna PUUV IgMb PUUV AVIc PUUV-RNA RT-PCR C3a TCC C3 C4 a b c d
ng/mL ng/mL g/L g/L
Ref. value
d d
0.5–1.5 0.12–0.42
10.3.2011
11.3.2011
20.4.2011
2.5.2011
Serum
Serum
EDTA-plasma
EDTA-plasma
640 Gran Pos 32 Neg >2000 499 0.45 0.06
1280 Gran Pos 64 Neg >2000 600 0.53 0.06
3.5.2011
9.5.2011
Serum
EDTA-plasma
2560 Gran/Diff Pos 8 392 245 1.53 0.14
160 160 1.36 0.12
>2000 894 1.31 0.11
18.4.2013 Serum 5120 Diffuse Neg 16
160 205 1.48 0.17
1837 410 1.71 0.27
Granular = acute, Diffuse = convalescent. l-capture EIA based on baculovirus-expressed nucleocapsid protein. IgG avidity IFA (ratio of low/high avidity IgG); TCC = terminal complement complex. For the complement activation products (C3a, TCC) no generally accepted reference values exist.
3. Clinical chemistry and hematological parameters The laboratory findings during the acute and recovery phase are presented in Table 2. They showed high hemoglobin (Hb) and low plasma albumin (reflecting severe capillary leakage), profound and long-lasting leukocytosis, thrombocytopenia, high plasma creatinine level (acute kidney injury), high ALT and TT-INR levels (liver failure), and low plasma levels of AT3 and fibrinogen, as well as high levels of FIDD (activated coagulation and fibrinolysis). Table 2 shows that on admission to hospital Hb was very high (216 g/L) after which at the acute phase of the disease the patient became anemic (Hb 78 g/L) and during the convalescent phase Hb normalized. Hb 174 g/L is probably the normal value of the patient. According to our experience, these Hb values are quite common in severe cases of PUUV infection. We have recently reported that the spleen is transiently enlarged during acute PUUV infection and that the enlargement correlates inversely with blood leukocyte count (Koskela et al., 2014). The present patient had earlier undergone splenectomy, which may have influenced the amount of leukocytosis. High serum levels of tPA (measured by ELISA, Sekisui Diagnostics) were found in the acute phase of the disease (41.4 ng/ml) while normal values were observed at full recovery (8.5 ng/ml). The strong upregulation of tPA suggestive of overt fibrinolysis has previously been reported to take place in acute PUUV infection (Laine et al., 2010). Extensive fibrinolysis might play a role in bleeding complications in PUUV infection by impairing normal
hemostasis. Plasma bradykinin level could not be measured as plasma had not been taken in the presence of protease inhibitors. Recently it was reported that hantavirus infection induces neutrophil extracellular traps (NETs) which results in elevated levels of circulating histones (Raftery et al., 2014). We therefore measured the serum levels of circulating histones by a dot-blot assay. 5-ll aliquots of serum were pipetted on nitrocellulose membrane, probed with rabbit polyclonal antibody detecting histone H3 (Abcam) in blocking buffer (1.5% milk in Tris–EDTA–NaCl–Tween (TENT)). After washing with TENT, the primary antibody was detected with IR800-conjugated anti-rabbit antibody (Li-cor) in blocking buffer. After washing, signal intensity was determined by Odyssey instrumentation (Li-cor). The signal intensity was approximately fivefold higher in the acute vs. recovery stages indicating elevated levels of histone H3 in the acute stage. Circulating histones are known to cause endothelial cell damage (Xu et al., 2009) and could thus be one factor contributing to the disease progression. 4. Complement activation Complement is activated in hantavirus infection (Sane et al., 2012) and many other inflammatory conditions (Meri, 2013). Plasma and serum C3 and C4 levels were measured by nephelometry. For both the C3a and the soluble terminal complement complex (TCC, SC5b-9) test either serum or plasma samples were available. To serum samples 0.01 M EDTA (final concentration)
Table 2 Biomarkers: clinical chemistry. Treatment in Intensive Care Unit Date of sample Test
Unit
Ref. value
Hemoglobin Platelets Leukocytes CRP Creatinine Albumin ALT P-TT-INR P-Fibrinogen P-FIDD P-AT3 t-PA Histones
g/L 109/L 109/L mg/L lmol/L g/L U/L
134–167 150–360 3.4–8.2 <10 60–100 36–48 10–70 0.9–1.2 2.0–4.0 0.0–0.5 80–120 5
g/L mg/L % ng/ml a
CRP = C-reactive protein, ALT = alanine aminotransferase. TT-INR = Thromboplastin time, FIDD = fibrinogen D-dimers. AT3 = antithrombin 3, t-PA = tissue plasminogen activator. a Dot-blot signal intensity.
10.3.2011
15.3.2011
22.3.2011
30.3.2011
18.4.2013
216 25 38.6 70 463 11 841 3.5 1.6 >4 30 41.4 4.8
105 59 28.2 32 210 8 2076 1.3 1.2 >4 25
78 253 12.0 22 81
120 452 8.1 2 49
174 420 8.3 2 62
178 1.2
104 1.2
40
25 8.5 1.0
A. Vaheri et al. / Antiviral Research 111 (2014) 23–25
was added prior to the analysis. C3a and TCC concentrations were measured using the MicroVue C3a Plus and MicroVue sC5b-9 EIA kits, respectively, according to the manufacturer’s instructions. During the acute phase low serum complement C3 (0.45 and 0.53 g/l) and C4 (0.06 g/l) levels were observed (Table 1). During follow-up the level of C3 normalized (to levels above 1.3 g/l). C4 levels increased (0.11–0.17 g/l) but remained still relatively low except for the sample taken on April 18th 2013 (0.27 g/l). The decreases in these key complement components suggest robust complement activation during the acute phase. Serum levels of C3a were, above the normal range, in the acute phase. Also, the levels of TCC were elevated (499 and 600 ng/ml). These point towards complement activation in conjunction with consumption of C3 and C4. It has to be noted, however, that because no EDTA (0.01 M) plasmas were available from the acute stage, the measurements were made from serum samples where EDTA was added only at the time of thawing of the samples. In serum samples further complement activation may occur in vitro, and the tendency for in vitro activation is higher if there is activation in the original sample. This is likely the explanation for the high serum levels of C3a and TCC (Table 1). The low levels of C3 and C4 nevertheless indicate a strong activation of complement and consumption at the time of the most severe clinical episode. Complement activation occurred concurrently with low levels of thrombocytes. The generation of complement activation products, especially of C5a and TCC, are known to lead to strong activation of platelets. The platelets thereby become adherent. They form aggregates and bind to endothelial cells, which together with leukocyte activation and aggregation leads to damage in small capillaries, which can cause a number of a respiratory distress syndrome and central nervous system disturbances. 5. Host genetics The relatively low levels of C4 suggested that the patient may also have a genetic deficiency in the polymorphic C4 gene. Therefore, both the copy numbers and allotypes of the C4A and C4B genes and proteins were determined by genomic real-time PCR and electrophoresis followed by immunofixation with polyclonal anti-C4-antibody, respectively (Paakkanen et al., 2012). No C4 gene deficiencies were observed. On the contrary the patient had three C4A genes and two C4B genes revealing that the C4 gene region had underwent gene rearrangements presenting duplicated C4A-like genes on the other chromosome. HLA-A, B, DRB1-typings were performed with commercial kit (Olerup SSP) showing an HLA-phenotype A⁄31,⁄32; B⁄15,⁄40; DRB1⁄08,⁄11. This haplotype is not common in the Finnish population (Wennerström et al., 2013) and no associations with viral infections have been previously reported. Surprisingly, almost exactly the same HLA-phenotype with C4 gene rearrangements causing C4B gene duplication in a family with a history of recurrent spontaneous abortions in early pregnancy has been previously described in the Finnish population (Jaatinen et al., 2002). Since no information of the patient’s family members was available it was not possible to confirm the MHC haplotypes and the origin of C4A. Surprisingly the patient was HLA-B⁄8 and DRB1⁄0301 negative. In our previous studies this haplotype has been associated with a severe course of PUUV infection (Mustonen et al., 1996; Mäkelä et al., 2002). In our preliminary study all seven patients with clinical shock had this HLA haplotype (Mustonen et al., 1996). 6. Concluding remarks The salient features in the pathophysiology of this patient were Strong complement activation, extensive fibrinolysis, uncommon
25
HLA haplotype, circulating histones, possible role of splenectomy and the recovery of very severely ill patient presumably by icatibant. It is well known that aspenic or splenectomized individuals are prone to life-threatening infections by encapsulated bacteria (Di Sabatino et al., 2011). We do not know any previous studies to show an association between the risk of viral infections and asplenism. Recently a patient with severe cytomegalovirus mononucleosis after splenectomy was described (Han et al., 2010). The authors suggested that the disease resulted from poor control of early viremia because of the lack of both splenic filtration and the typical brisk IgM response. Acknowledgments We thank Jussi Hepojoki and Tarja Sironen for discussion and Kirsi Aaltonen and Mira Utriainen for technical assistance. This work was supported by grants from Sigrid Jusélius Foundation, Academy of Finland, Magnus Ehrnrooth Foundation, European Commission Project QLK2-CT-2002-01358, The Competitive State Research Funding of the Expert Responsibility Area of Tampere University Hospital, and Helsinki University Hospital, Hospital district of Helsinki and Uusimaa (TYH-2011305) Research Funds. References Antonen, J., Leppänen, I., Tenhunen, J., Arvola, P., Mäkelä, S., Vaheri, A., Mustonen, J., 2013. A severe case of Puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant. Scand. J. Infect. Dis. 45, 494–496. Di Sabatino, A., Carsetti, R., Corazza, G.R., 2011. Post-splenectomy and hyposplenic states. Lancet 378, 86–97. Han, X.Y., Hellerstedt, B.A., Koller, C.A., 2010. Postsplenectomy cytomegalovirus mononucleosis is a distinct clinicopathologic syndrome. Am. J. Med. Sci. 33, 395–399. Jaatinen, T., Eholuoto, M., Laitinen, T., Lokki, M.L., 2002. Characterization of a de novo conversion in human complement C4 gene producing a C4B5-like protein. J. Immunol. 168, 5652–5658. Koskela, S.M., Laine, O.K., Paakkala, A.S., Mäkelä, S.M., Mustonen, J.T., 2014. Spleen enlargement is a common finding in acute Puumala hantavirus infection and it does not associate with thrombocytopenia. Scand. J. Infect. Dis. (in press). Laine, O., Mäkelä, S., Mustonen, J., Huhtala, H., Szanto, T., Vaheri, A., Lassila, R., Joutsi-Korhonen, L., 2010. Enhanced thrombin formation and fibrinolysis during acute Puumala hantavirus infection. Thromb. Res. 126, 154–158. Mäkelä, S., Mustonen, J., Ala-Houhala, I., Hurme, M., Partanen, J., Vapalahti, O., Vaheri, A., Pasternack, A., 2002. Human leukocyte antigen-B8-DR3 is a more important risk factor for severe Puumala hantavirus infection than the tumor necrosis factor-a(–308 G/A polymorphism. J. Infect. Dis. 186, 843–846. Meri, S., 2013. Complement activation in diseases presenting with thrombotic microangiopathy. Eur. J. Int. Med. 24, 496–502. Mustonen, J., Mäkelä, S., Outinen, T., Laine, T., Jylhävä, J., Arstila, P.T., Hurme, M., Vaheri, A., 2013. The pathogenesis of nephropathia epidemica: new knowledge and unanswered questions. Antiviral Res. 100, 589–604. Mustonen, J., Partanen, J., Kanerva, M., Pietilä, K., Vapalahti, O., Pasternack, A., Vaheri, A., 1996. Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus. Kidney Int. 49, 217–221. Paakkanen, R., Vauhkonen, H., Eronen, K.T., Järvinen, A., Seppänen, M., Lokki, M.L., 2012. Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain reaction. PLoS One 7 (6), e38813. http://dx.doi.org/10.1371/journal.pone.0038813. Raftery, M.J., Lalwani, P., Krautkrämer, E., Peters, T., Scharffetter-Kochanek, K., Krüger, R., Hofmann, J., Seeger, K., Krüger, D.H., Schönrich, G., 2014. b2 integrin mediates hantavirus-induced release of neutrophil extracellular traps, J. Exp. Med. Sane, J., Laine, O., Mäkelä, S., Paakkala, A., Jarva, H., Mustonen, J., Vapalahti, O., Meri, S., Vaheri, A., 2012. Complement activation in Puumala hantavirus infection correlates with disease severity. Ann. Med. 44, 468–475. Vaheri, A., Vapalahti, O., Plyusnin, A., 2008. How to diagnose hantavirus infections and detect them in rodents and insectivores. Rev. Med. Virol. 8, 277–288. Wennerström, A., Vlachopoulou, E., Lahtela, L.E., Paakkanen, R., Eronen, K.T., Seppänen, M., Lokki, M.L., 2013. Diversity of extended HLA-DRB1 haplotypes in the Finnish population. PLoS One 8 (11), e79690. http://dx.doi.org/10.1371/ journal.pone.0079690. Xu, J., Zhang, X., Pelayo, R., Monestier, M., Ammollo, C.T., Semeraro, F., Taylor, F.B., Esmon, N.L., Lupu, F., Esmon, C.T., 2009. Extracellular histones are major mediators of death in sepsis. Nat. Med. 15, 1318–1321.
Contents lists available at ScienceDirect
Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
Short Communication
Pathophysiology of a severe case of Puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant Antti Vaheri a,b,⇑, Tomas Strandin a, Anne J. Jääskeläinen a,b, Olli Vapalahti a,b,c, Hanna Jarva a,d, Marja-Liisa Lokki a, Jaakko Antonen e, Ilona Leppänen e, Satu Mäkelä e,f, Seppo Meri a,d, Jukka Mustonen e,f a
Haartman Institute, University of Helsinki, Finland Department of Virology and Immunology, Helsinki University Central Hospital Laboratory, Finland Department of Veterinary Biosciences, University of Helsinki, Finland d Immunobiology Research Program, University of Helsinki, Finland e Tampere University Hospital, Finland f School of Medicine, University of Tampere, Finland b c
a r t i c l e
i n f o
Article history: Received 30 May 2014 Revised 11 August 2014 Accepted 14 August 2014 Available online 3 September 2014 Keywords: Hantavirus Pathophysiology Capillary leakage Complement activation Bradykinin Icatibant
a b s t r a c t We recently described a patient with very severe Puumala hantavirus infection manifested by capillary leakage syndrome and shock. He was successfully treated with the bradykinin receptor antagonist, icatibant (Antonen et al., 2013). Here we report analysis of the pathophysiology which indicated pronounced complement activation, prolonged leukocytosis, extensive fibrinolysis, circulating histones, and defects in liver function. The patient had an uncommon HLA-phenotype, which may have contributed to the severe course of the disease. Ó 2014 Elsevier B.V. All rights reserved.
Introduction The patient was a previously healthy 37-year-old Finnish male, except he had undergone 8 years earlier splenectomy due to congenital spherocytosis. He was admitted to hospital on March 10th 2011 due to acute Puumala virus (PUUV) infection. He suffered from a severe capillary leakage, shock, and multiorgan failure including kidney, liver and respiratory failure (Antonen et al., 2013). He was treated in the intensive care unit (ICU) until 23rd March 2011 and the total hospital care lasted until April 4th 2011. The treatment in the ICU included mechanical ventilation, continuous renal replacement therapy, massive fluid replacement, vasoactive drugs, inhaled nitric oxide, corticosteroids and ceftriaxone. He also got one subcutaneous dose of icatibant. The rationale was that a central pathophysiological aberration in hantavirus ⇑ Corresponding author at: Department of Virology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland. Tel.: +358 50 448 2850; fax: +358 2941 26491. E-mail address: antti.vaheri@helsinki.fi (A. Vaheri). http://dx.doi.org/10.1016/j.antiviral.2014.08.007 0166-3542/Ó 2014 Elsevier B.V. All rights reserved.
infections is increased capillary permeability. Bradykinin promotes permeability in many pathological conditions. Icatibant is a selective antagonist of the bradykinin type 2 receptor, which is indicated for the treatment of acute episodes of hereditary angioedema. During the next 24 h after dosing of icatibant the condition of the patient stabilized, followed by gradual improvement and a full recovery (Antonen et al., 2013).
2. Virological parameters PUUV is the only hantaviral pathogen in Finland and is carried by bank voles (Mustonen et al., 2013). The data in Table 1 confirm that the patient had acute PUUV infection: IgM antibodies, typical granular fluorescence pattern in immunofluorescence antibody test (IFAT) and high IgG-antibody avidity ratio (Vaheri et al., 2008). No PUUV RNA was detected in the serum. The patient had no antibodies to the following zoonosis-related viruses: cowpox, Ljungan, lymphocytic choriomeningitis or tick-borne encephalitis virus and thus no coinfections with these viruses were detected to explain the unusually severe disease.
24
A. Vaheri et al. / Antiviral Research 111 (2014) 23–25
Table 1 Biomarkers: virology and complement. Date of sample Test
Unit
PUUV IgG IFA titer PUUV IgG IFA patterna PUUV IgMb PUUV AVIc PUUV-RNA RT-PCR C3a TCC C3 C4 a b c d
ng/mL ng/mL g/L g/L
Ref. value
d d
0.5–1.5 0.12–0.42
10.3.2011
11.3.2011
20.4.2011
2.5.2011
Serum
Serum
EDTA-plasma
EDTA-plasma
640 Gran Pos 32 Neg >2000 499 0.45 0.06
1280 Gran Pos 64 Neg >2000 600 0.53 0.06
3.5.2011
9.5.2011
Serum
EDTA-plasma
2560 Gran/Diff Pos 8 392 245 1.53 0.14
160 160 1.36 0.12
>2000 894 1.31 0.11
18.4.2013 Serum 5120 Diffuse Neg 16
160 205 1.48 0.17
1837 410 1.71 0.27
Granular = acute, Diffuse = convalescent. l-capture EIA based on baculovirus-expressed nucleocapsid protein. IgG avidity IFA (ratio of low/high avidity IgG); TCC = terminal complement complex. For the complement activation products (C3a, TCC) no generally accepted reference values exist.
3. Clinical chemistry and hematological parameters The laboratory findings during the acute and recovery phase are presented in Table 2. They showed high hemoglobin (Hb) and low plasma albumin (reflecting severe capillary leakage), profound and long-lasting leukocytosis, thrombocytopenia, high plasma creatinine level (acute kidney injury), high ALT and TT-INR levels (liver failure), and low plasma levels of AT3 and fibrinogen, as well as high levels of FIDD (activated coagulation and fibrinolysis). Table 2 shows that on admission to hospital Hb was very high (216 g/L) after which at the acute phase of the disease the patient became anemic (Hb 78 g/L) and during the convalescent phase Hb normalized. Hb 174 g/L is probably the normal value of the patient. According to our experience, these Hb values are quite common in severe cases of PUUV infection. We have recently reported that the spleen is transiently enlarged during acute PUUV infection and that the enlargement correlates inversely with blood leukocyte count (Koskela et al., 2014). The present patient had earlier undergone splenectomy, which may have influenced the amount of leukocytosis. High serum levels of tPA (measured by ELISA, Sekisui Diagnostics) were found in the acute phase of the disease (41.4 ng/ml) while normal values were observed at full recovery (8.5 ng/ml). The strong upregulation of tPA suggestive of overt fibrinolysis has previously been reported to take place in acute PUUV infection (Laine et al., 2010). Extensive fibrinolysis might play a role in bleeding complications in PUUV infection by impairing normal
hemostasis. Plasma bradykinin level could not be measured as plasma had not been taken in the presence of protease inhibitors. Recently it was reported that hantavirus infection induces neutrophil extracellular traps (NETs) which results in elevated levels of circulating histones (Raftery et al., 2014). We therefore measured the serum levels of circulating histones by a dot-blot assay. 5-ll aliquots of serum were pipetted on nitrocellulose membrane, probed with rabbit polyclonal antibody detecting histone H3 (Abcam) in blocking buffer (1.5% milk in Tris–EDTA–NaCl–Tween (TENT)). After washing with TENT, the primary antibody was detected with IR800-conjugated anti-rabbit antibody (Li-cor) in blocking buffer. After washing, signal intensity was determined by Odyssey instrumentation (Li-cor). The signal intensity was approximately fivefold higher in the acute vs. recovery stages indicating elevated levels of histone H3 in the acute stage. Circulating histones are known to cause endothelial cell damage (Xu et al., 2009) and could thus be one factor contributing to the disease progression. 4. Complement activation Complement is activated in hantavirus infection (Sane et al., 2012) and many other inflammatory conditions (Meri, 2013). Plasma and serum C3 and C4 levels were measured by nephelometry. For both the C3a and the soluble terminal complement complex (TCC, SC5b-9) test either serum or plasma samples were available. To serum samples 0.01 M EDTA (final concentration)
Table 2 Biomarkers: clinical chemistry. Treatment in Intensive Care Unit Date of sample Test
Unit
Ref. value
Hemoglobin Platelets Leukocytes CRP Creatinine Albumin ALT P-TT-INR P-Fibrinogen P-FIDD P-AT3 t-PA Histones
g/L 109/L 109/L mg/L lmol/L g/L U/L
134–167 150–360 3.4–8.2 <10 60–100 36–48 10–70 0.9–1.2 2.0–4.0 0.0–0.5 80–120 5
g/L mg/L % ng/ml a
CRP = C-reactive protein, ALT = alanine aminotransferase. TT-INR = Thromboplastin time, FIDD = fibrinogen D-dimers. AT3 = antithrombin 3, t-PA = tissue plasminogen activator. a Dot-blot signal intensity.
10.3.2011
15.3.2011
22.3.2011
30.3.2011
18.4.2013
216 25 38.6 70 463 11 841 3.5 1.6 >4 30 41.4 4.8
105 59 28.2 32 210 8 2076 1.3 1.2 >4 25
78 253 12.0 22 81
120 452 8.1 2 49
174 420 8.3 2 62
178 1.2
104 1.2
40
25 8.5 1.0
A. Vaheri et al. / Antiviral Research 111 (2014) 23–25
was added prior to the analysis. C3a and TCC concentrations were measured using the MicroVue C3a Plus and MicroVue sC5b-9 EIA kits, respectively, according to the manufacturer’s instructions. During the acute phase low serum complement C3 (0.45 and 0.53 g/l) and C4 (0.06 g/l) levels were observed (Table 1). During follow-up the level of C3 normalized (to levels above 1.3 g/l). C4 levels increased (0.11–0.17 g/l) but remained still relatively low except for the sample taken on April 18th 2013 (0.27 g/l). The decreases in these key complement components suggest robust complement activation during the acute phase. Serum levels of C3a were, above the normal range, in the acute phase. Also, the levels of TCC were elevated (499 and 600 ng/ml). These point towards complement activation in conjunction with consumption of C3 and C4. It has to be noted, however, that because no EDTA (0.01 M) plasmas were available from the acute stage, the measurements were made from serum samples where EDTA was added only at the time of thawing of the samples. In serum samples further complement activation may occur in vitro, and the tendency for in vitro activation is higher if there is activation in the original sample. This is likely the explanation for the high serum levels of C3a and TCC (Table 1). The low levels of C3 and C4 nevertheless indicate a strong activation of complement and consumption at the time of the most severe clinical episode. Complement activation occurred concurrently with low levels of thrombocytes. The generation of complement activation products, especially of C5a and TCC, are known to lead to strong activation of platelets. The platelets thereby become adherent. They form aggregates and bind to endothelial cells, which together with leukocyte activation and aggregation leads to damage in small capillaries, which can cause a number of a respiratory distress syndrome and central nervous system disturbances. 5. Host genetics The relatively low levels of C4 suggested that the patient may also have a genetic deficiency in the polymorphic C4 gene. Therefore, both the copy numbers and allotypes of the C4A and C4B genes and proteins were determined by genomic real-time PCR and electrophoresis followed by immunofixation with polyclonal anti-C4-antibody, respectively (Paakkanen et al., 2012). No C4 gene deficiencies were observed. On the contrary the patient had three C4A genes and two C4B genes revealing that the C4 gene region had underwent gene rearrangements presenting duplicated C4A-like genes on the other chromosome. HLA-A, B, DRB1-typings were performed with commercial kit (Olerup SSP) showing an HLA-phenotype A⁄31,⁄32; B⁄15,⁄40; DRB1⁄08,⁄11. This haplotype is not common in the Finnish population (Wennerström et al., 2013) and no associations with viral infections have been previously reported. Surprisingly, almost exactly the same HLA-phenotype with C4 gene rearrangements causing C4B gene duplication in a family with a history of recurrent spontaneous abortions in early pregnancy has been previously described in the Finnish population (Jaatinen et al., 2002). Since no information of the patient’s family members was available it was not possible to confirm the MHC haplotypes and the origin of C4A. Surprisingly the patient was HLA-B⁄8 and DRB1⁄0301 negative. In our previous studies this haplotype has been associated with a severe course of PUUV infection (Mustonen et al., 1996; Mäkelä et al., 2002). In our preliminary study all seven patients with clinical shock had this HLA haplotype (Mustonen et al., 1996). 6. Concluding remarks The salient features in the pathophysiology of this patient were Strong complement activation, extensive fibrinolysis, uncommon
25
HLA haplotype, circulating histones, possible role of splenectomy and the recovery of very severely ill patient presumably by icatibant. It is well known that aspenic or splenectomized individuals are prone to life-threatening infections by encapsulated bacteria (Di Sabatino et al., 2011). We do not know any previous studies to show an association between the risk of viral infections and asplenism. Recently a patient with severe cytomegalovirus mononucleosis after splenectomy was described (Han et al., 2010). The authors suggested that the disease resulted from poor control of early viremia because of the lack of both splenic filtration and the typical brisk IgM response. Acknowledgments We thank Jussi Hepojoki and Tarja Sironen for discussion and Kirsi Aaltonen and Mira Utriainen for technical assistance. This work was supported by grants from Sigrid Jusélius Foundation, Academy of Finland, Magnus Ehrnrooth Foundation, European Commission Project QLK2-CT-2002-01358, The Competitive State Research Funding of the Expert Responsibility Area of Tampere University Hospital, and Helsinki University Hospital, Hospital district of Helsinki and Uusimaa (TYH-2011305) Research Funds. References Antonen, J., Leppänen, I., Tenhunen, J., Arvola, P., Mäkelä, S., Vaheri, A., Mustonen, J., 2013. A severe case of Puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant. Scand. J. Infect. Dis. 45, 494–496. Di Sabatino, A., Carsetti, R., Corazza, G.R., 2011. Post-splenectomy and hyposplenic states. Lancet 378, 86–97. Han, X.Y., Hellerstedt, B.A., Koller, C.A., 2010. Postsplenectomy cytomegalovirus mononucleosis is a distinct clinicopathologic syndrome. Am. J. Med. Sci. 33, 395–399. Jaatinen, T., Eholuoto, M., Laitinen, T., Lokki, M.L., 2002. Characterization of a de novo conversion in human complement C4 gene producing a C4B5-like protein. J. Immunol. 168, 5652–5658. Koskela, S.M., Laine, O.K., Paakkala, A.S., Mäkelä, S.M., Mustonen, J.T., 2014. Spleen enlargement is a common finding in acute Puumala hantavirus infection and it does not associate with thrombocytopenia. Scand. J. Infect. Dis. (in press). Laine, O., Mäkelä, S., Mustonen, J., Huhtala, H., Szanto, T., Vaheri, A., Lassila, R., Joutsi-Korhonen, L., 2010. Enhanced thrombin formation and fibrinolysis during acute Puumala hantavirus infection. Thromb. Res. 126, 154–158. Mäkelä, S., Mustonen, J., Ala-Houhala, I., Hurme, M., Partanen, J., Vapalahti, O., Vaheri, A., Pasternack, A., 2002. Human leukocyte antigen-B8-DR3 is a more important risk factor for severe Puumala hantavirus infection than the tumor necrosis factor-a(–308 G/A polymorphism. J. Infect. Dis. 186, 843–846. Meri, S., 2013. Complement activation in diseases presenting with thrombotic microangiopathy. Eur. J. Int. Med. 24, 496–502. Mustonen, J., Mäkelä, S., Outinen, T., Laine, T., Jylhävä, J., Arstila, P.T., Hurme, M., Vaheri, A., 2013. The pathogenesis of nephropathia epidemica: new knowledge and unanswered questions. Antiviral Res. 100, 589–604. Mustonen, J., Partanen, J., Kanerva, M., Pietilä, K., Vapalahti, O., Pasternack, A., Vaheri, A., 1996. Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus. Kidney Int. 49, 217–221. Paakkanen, R., Vauhkonen, H., Eronen, K.T., Järvinen, A., Seppänen, M., Lokki, M.L., 2012. Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain reaction. PLoS One 7 (6), e38813. http://dx.doi.org/10.1371/journal.pone.0038813. Raftery, M.J., Lalwani, P., Krautkrämer, E., Peters, T., Scharffetter-Kochanek, K., Krüger, R., Hofmann, J., Seeger, K., Krüger, D.H., Schönrich, G., 2014. b2 integrin mediates hantavirus-induced release of neutrophil extracellular traps, J. Exp. Med. Sane, J., Laine, O., Mäkelä, S., Paakkala, A., Jarva, H., Mustonen, J., Vapalahti, O., Meri, S., Vaheri, A., 2012. Complement activation in Puumala hantavirus infection correlates with disease severity. Ann. Med. 44, 468–475. Vaheri, A., Vapalahti, O., Plyusnin, A., 2008. How to diagnose hantavirus infections and detect them in rodents and insectivores. Rev. Med. Virol. 8, 277–288. Wennerström, A., Vlachopoulou, E., Lahtela, L.E., Paakkanen, R., Eronen, K.T., Seppänen, M., Lokki, M.L., 2013. Diversity of extended HLA-DRB1 haplotypes in the Finnish population. PLoS One 8 (11), e79690. http://dx.doi.org/10.1371/ journal.pone.0079690. Xu, J., Zhang, X., Pelayo, R., Monestier, M., Ammollo, C.T., Semeraro, F., Taylor, F.B., Esmon, N.L., Lupu, F., Esmon, C.T., 2009. Extracellular histones are major mediators of death in sepsis. Nat. Med. 15, 1318–1321.