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Table of Contents
Vol. 75, No. 2, 2008
Issue release date: March 2008
Respiration 2008;75:155–157
(DOI:10.1159/000097495)

Absence of Human Herpesvirus 8 DNA Sequences in Lung Biopsies from Israeli Patients with Pulmonary Arterial Hypertension

Bendayan D.a · Sarid R.c · Cohen A.c · Shitrit D.a · Shechtman I.b · Kramer M.R.a
aPulmonary Institute, and bPathology Institute, Rabin Medical Center, Petah Tikvah, and cFaculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
email Corresponding Author

Abstract

Background:Pulmonary hypertension is a severe pulmonary vascular disease leading to rapid deterioration and death. Histological and clinical evidence suggests that smooth muscle proliferation is part of the pathogenesis of the disease. Human herpesvirus 8 (HHV-8) is a γ-herpesvirus that is implicated in malignancies and in Kaposi’s sarcoma. Recently, the association of HHV-8 with idiopathic pulmonary arterial hypertension (PAH) has been found. Objective: The aim of this study was to investigate the presence of HHV-8 in the lung tissue of Israeli patients with PAH. Method: The presence of HHV-8 sequences was investigated by polymerase chain reaction examination in 6 biopsies of patients with pulmonary hypertension. Three patients had idiopathic pulmonary hypertension, 2 patients pulmonary venoocclusive disease, and 1 patient pulmonary hypertension associated with mixed connective tissue disease. Result: We did not find any association between HHV-8 and PAH in these Israeli patients, as all the samples were negative for polymerase chain reaction. Conclusion: Our findings, together with the epidemiological data of HHV-8 prevalence and incidence rates of Kaposi’s sarcoma and PAH in Israel, provide further evidence which argues against an association between HHV-8 infection and PAH.


 Outline


 goto top of outline Key Words

  • Human herpesvirus 8
  • Pulmonary arterial hypertension

 goto top of outline Abstract

Background:Pulmonary hypertension is a severe pulmonary vascular disease leading to rapid deterioration and death. Histological and clinical evidence suggests that smooth muscle proliferation is part of the pathogenesis of the disease. Human herpesvirus 8 (HHV-8) is a γ-herpesvirus that is implicated in malignancies and in Kaposi’s sarcoma. Recently, the association of HHV-8 with idiopathic pulmonary arterial hypertension (PAH) has been found. Objective: The aim of this study was to investigate the presence of HHV-8 in the lung tissue of Israeli patients with PAH. Method: The presence of HHV-8 sequences was investigated by polymerase chain reaction examination in 6 biopsies of patients with pulmonary hypertension. Three patients had idiopathic pulmonary hypertension, 2 patients pulmonary venoocclusive disease, and 1 patient pulmonary hypertension associated with mixed connective tissue disease. Result: We did not find any association between HHV-8 and PAH in these Israeli patients, as all the samples were negative for polymerase chain reaction. Conclusion: Our findings, together with the epidemiological data of HHV-8 prevalence and incidence rates of Kaposi’s sarcoma and PAH in Israel, provide further evidence which argues against an association between HHV-8 infection and PAH.

Copyright © 2007 S. Karger AG, Basel


goto top of outline Introduction

Human herpesvirus 8 (HHV-8), also named Kaposi’s sarcoma (KS)-associated herpesvirus, was initially identified in KS, a complex multifocal neoplasm characterized by proliferation of spindle-like cells, substantial angiogenesis, and inflammation [1]. Subsequently, an etiologic association between HHV-8 and all forms of KS has been well established. HHV-8 is also implicated in the majority of primary effusion lymphoma (PEL) cases and in approximately 50% of multicentric Castleman’s disease (MCD) cases. KS is the most common AIDS-related cancer, while PEL and MCD are rare conditions that are mainly associated with HIV-1 infection. A number of studies reported elevated the prevalence of HHV-8 infection among HIV-1-infected individuals [2, 3].

Idiopathic pulmonary arterial hypertension (IPAH) is a severe rare pulmonary vascular disease leading to rapid deterioration and death [4]. Histological examinations of lung biopsies from IPAH patients demonstrate vascular lesions in the lung and proliferation of endothelial and smooth muscle cells. The etiology of the disease is unknown in most cases, while a subgroup of patients with familial IPAH harbors germline mutations of bone morphogenetic protein receptor 2 [5]. An association between HHV-8 infection and IPAH has recently been suggested [6, 7], while subsequent studies failed to confirm this notion.

In the present study, we investigated the presence of HHV-8 in biopsies from Israeli patients with PAH.

 

goto top of outline Patients and Methods

goto top of outline Patients

During 2003–2005, we obtained formalin-fixed paraffin-embedded biopsies from 6 patients with PAH. Four lung biopsies were obtained from 4 patients – 3 after heart-lung transplantation and 1 after autopsy. Transbronchial biopsy samples were obtained from an additional 2 patients. Three patients were diagnosed with IPAH, 2 had pulmonary venoocclusive disease, and 1 had pulmonary hypertension associated with mixed connective tissue disease. All patients were living in Israel; 5 were Jewish and 1 was an Arab (patient 5). Patients 1–4 were from East European origin and patient 6 was born in Ethiopia. The characteristics of the patients are described in table 1.

TAB01
Table 1. Patient characteristics

goto top of outline Methods

DNA was extracted from the 6 pulmonary samples and examined for the presence of HHV-8 sequences by polymerase chain reaction (PCR).

Tissue sections were deparaffinized, and the remaining pel- let was resuspended, incubated overnight at 55°C in lysis buffer (50 mM Tris, pH 8.5, 1 mM EDTA, 0.5% Tween-20 and 200 μg/ml proteinase K) and then incubated for 10 min at 95°C to inactivate protease. Aliquots were used for nested PCR amplification targeting two HHV-8 genes – ORF 26 and ORF K1. The first primer set, 5′-TCCGTGTTGTCTACGTCCA-3′ and 5′-AGCCGAAAGGATTCCACCAT-3′, was designed for the amplification of the KS330223 DNA sequence of HHV-8, as described, and a second primer set, 5′-ACGATATGTGCGCCCCATAA-3′ and 5′-TGTGCTCGAATCCAACGGAT-3′, was used for nested PCR amplification. The K1 amplification employed 5′-ATGTTCCTGTATGTTGTCTGC-3′ and 5′-TCAGTACCAATCCACTGGTT-3′ and a second primer set, 5′-TATGTTGTCTGCAGTCTGGCG-3′ and 5′-ATCCACTGGTTGCGTATAGTC-3′, for nested PCR amplification. All samples were also amplified with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer sets 5′-ACCACAGTCCATGCCATCAC and 5′-TTTTCTAGACGGCAGGTCAG-3′, and for the nest PCR set, with 5′-TGCCACCCAGA- AGACTGTGGA-3′ and 5′-ACCACTGACACGTTGGCAGT-3′. Negative controls including a negative biopsy specimen and buffer controls were simultaneously assayed to monitor for PCR contamination. As a positive control, we used high-molecular DNA extracted from an HHV-8-infected cell line. Products of nested PCR reactions were visualized on 2% agarose gel containing ethidium bromide.

 

goto top of outline Result

All samples tested were negative for ORF 26 and ORF K1 HHV-8 genes, while their integrity and suitability for amplification was confirmed by amplification of the cellular gene GAPDH.

 

goto top of outline Discussion

The association of HHV-8 infection with various pathological conditions has been widely investigated using genomic, histological and serological approaches. However, most studies could not substantiate the association of HHV-8 with other human diseases apart from KS, PEL and MCD [2]. One research group has recently identified the genomic sequences of HHV-8 and the expression of the latency-associated nuclear antigen 1 encoded by HHV-8 in 10/16 (62%) lung biopsies from IPAH; 1 of the HHV-8-positive patients was also diagnosed with MCD [6, 7]. Only 1/14 biopsies from a patient with secondary pulmonary hypertension had PCR evidence of HHV-8 infection, with no detection of latency-associated nuclear antigen 1 expression [6]. Another case of HHV-8 associated with PAH was described in an HIV patient with MCD. HHV-8 was positive using a lytic immunofluorescence assay, and there was a complete reversibility following antiretroviral and anti-inflammatory therapy in association with epoprostenol [8]. In contrast, others failed to confirm the detection of HHV-8 in 10 IPAH Japanese patients [9] and in 26 IPAH German patients [10]. Furthermore, comparative serological studies among German, French, American and Italian patients with pulmonary hypertension did not find any evidence supporting a relationship between HHV-8 infection and IPAH [11,12,13,14]. Thus, our failure to detect the HHV-8 genome in biopsies from patients with IPAH disagrees with early results reported by one research group, but is in line with proceeding reports.

Further support for the lack of an association between HHV-8 and IPAH is obtained from epidemiological records. As the prevalence of HHV-8 infection varies between geographic regions, the involvement of HHV-8 with pathological conditions may happen more frequently in populations with a high prevalence of HHV-8. This has been extensively demonstrated for KS; however, no ethnic predisposition for IPAH was recorded by the National Institutes of Health registry, disproving an association between HHV-8 and IPAH. This is further strengthened by data collected from Israel. An estimated IPAH incidence rate of 1.4 new cases per million population per year was reported in a retrospective study [15]. This study found a relatively higher incidence rate among the immigrants from Europe and North America as compared with the general population. However, in Israel, the prevalence of HHV-8 infection [16] and the incidence rate of KS [17] are elevated among immigrants from Morocco and lower among immigrants from North America and Europe. Moreover, IPAH incidence rates in Israel are similar to those reported in the United States and in Europe, while KS incidence rates and HHV-8 prevalence are relatively higher in Israel [18]. Collectively, our present findings, together with several other reports and epidemiological records, suggest that there is no association between HHV-8 infection and IPAH.


 goto top of outline References
  1. Chang Y, Cesarman E, Pessin MS, et al: Identification of herpesvirus like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 1994;266:1865–1869.
  2. Cohen A, Wolf DG, Guttman-Yassky E, Sarid D: Kaposi sarcoma associated with herpesvirus: clinical, diagnostic and epidemiologic aspects. Crit Rev Clin Lab Sci 2005;42:101–153.
  3. Belec L, Mohamed AS, Authier FJ, et al: Human herpesvirus 8 infection in patients with POEMS syndrome-associated multicentric Castleman’s disease. Blood 1999;93:3643–3653.
  4. Rubin LJ: Primary pulmonary hypertension. N Engl J Med 1997;336:111–117.
  5. Galie N, Manes A, Uguccioni L, et al: Primary pulmonary hypertension: insights into pathogenesis from epidemiology. Chest 1998;114:184s–194s.
  6. Cool CD, Rai PR, Yeager ME, et al: Expression of human herpesvirus 8 in primary pulmonary hypertension. N Engl J Med 2003;349:1113–1122.
  7. Bull T, Cool CD, Serls AE, et al: Primary pulmonary hypertension, Castleman’s disease and human herpesvirus-8. Eur Respir J 2003;22:403–407.
  8. Montani D, Achouh L, Marsellin AG, et al: Reversibility of pulmonary arterial hypertension in HIV/HHV8 associated Castelman’s disease. Eur Respir J 2005;26:969–972.
  9. Katano H, Ito K, Shibuya K, et al: Lack of human herpesvirus 8 infection in lungs of Japanese patients with primary pulmonary hypertension. J Infect Dis 2005;191:743–745.
  10. Henke-Gendo C, Mengel M, Hoeper M, et al: Absence of Kaposi’s sarcoma-associated herpesvirus in patients with pulmonary arterial hypertension. Am J Respir Crit Care Med 2005;172:1581–1585.
  11. Henke-Gendo C, Schulz TF, Hoeper MM: HHV-8 in pulmonary hypertension. N Engl J Med 2004;350:194–195.
  12. Laney AS, De Marco T, Peters JS, et al: Kaposi sarcoma-associated herpesvirus and primary and secondary pulmonary hypertension. Chest 2005;127:762–767.
  13. Montani D, Marsellin AG, Sitbon O, et al: Human herpes virus 8 in HIV and non-HIV infected patients with pulmonary arterial hypertension in France. AIDS 2005;19:1239–1240.
  14. Nicastri E: Human herpesvirus 8 and pulmonary hypertension. Emerg Infect Dis 2005;11:1480–1482.
  15. Appelbaum L, Yigla M, Bendayan D, et al: Primary pulmonary hypertension in Israel: a national survey. Chest 2001;119:1801–1806.
  16. Davidovici B, Karakis I, Bourboulia D, et al: Seroepidemiology and molecular epidemiology of Kaposi’s sarcoma-associated herpesvirus among Jewish population groups in Israel. J Natl Cancer Inst 2001;93:194–202.
  17. Guttman-Yassky E, Bar-Chana M, Yukelson A, et al: Epidemiology of classic Kaposi’s sarcoma in the Israeli Jewish population between 1960 and 1998. Br J Cancer 2003;89:1657–1660.
  18. Iscovich J, Boffetta P, Franceschi S, et al: Classic Kaposi sarcoma: epidemiology and risk factors. Cancer 2000;88:500–517.

 goto top of outline Author Contacts

M.R. Kramer
Pulmonary Institute
Rabin Medical Center, Beilinson Campus
Petah Tikva 49100 (Israel)
Tel. +972 3 937 7221, Fax +972 3 924 2091, E-Mail daniellbd@hotmail.com


 goto top of outline Article Information

Received: May 18, 2006
Accepted after revision: September 9, 2006
Published online: November 23, 2006
Number of Print Pages : 3
Number of Figures : 0, Number of Tables : 1, Number of References : 18


 goto top of outline Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 75, No. 2, Year 2008 (Cover Date: March 2008)

Journal Editor: Bolliger, C.T. (Cape Town)
ISSN: 0025–7931 (Print), eISSN: 1423–0356 (Online)

For additional information: http://www.karger.com/RES


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Background:Pulmonary hypertension is a severe pulmonary vascular disease leading to rapid deterioration and death. Histological and clinical evidence suggests that smooth muscle proliferation is part of the pathogenesis of the disease. Human herpesvirus 8 (HHV-8) is a γ-herpesvirus that is implicated in malignancies and in Kaposi’s sarcoma. Recently, the association of HHV-8 with idiopathic pulmonary arterial hypertension (PAH) has been found. Objective: The aim of this study was to investigate the presence of HHV-8 in the lung tissue of Israeli patients with PAH. Method: The presence of HHV-8 sequences was investigated by polymerase chain reaction examination in 6 biopsies of patients with pulmonary hypertension. Three patients had idiopathic pulmonary hypertension, 2 patients pulmonary venoocclusive disease, and 1 patient pulmonary hypertension associated with mixed connective tissue disease. Result: We did not find any association between HHV-8 and PAH in these Israeli patients, as all the samples were negative for polymerase chain reaction. Conclusion: Our findings, together with the epidemiological data of HHV-8 prevalence and incidence rates of Kaposi’s sarcoma and PAH in Israel, provide further evidence which argues against an association between HHV-8 infection and PAH.



 goto top of outline Author Contacts

M.R. Kramer
Pulmonary Institute
Rabin Medical Center, Beilinson Campus
Petah Tikva 49100 (Israel)
Tel. +972 3 937 7221, Fax +972 3 924 2091, E-Mail daniellbd@hotmail.com


 goto top of outline Article Information

Received: May 18, 2006
Accepted after revision: September 9, 2006
Published online: November 23, 2006
Number of Print Pages : 3
Number of Figures : 0, Number of Tables : 1, Number of References : 18


 goto top of outline Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 75, No. 2, Year 2008 (Cover Date: March 2008)

Journal Editor: Bolliger, C.T. (Cape Town)
ISSN: 0025–7931 (Print), eISSN: 1423–0356 (Online)

For additional information: http://www.karger.com/RES


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Chang Y, Cesarman E, Pessin MS, et al: Identification of herpesvirus like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 1994;266:1865–1869.
  2. Cohen A, Wolf DG, Guttman-Yassky E, Sarid D: Kaposi sarcoma associated with herpesvirus: clinical, diagnostic and epidemiologic aspects. Crit Rev Clin Lab Sci 2005;42:101–153.
  3. Belec L, Mohamed AS, Authier FJ, et al: Human herpesvirus 8 infection in patients with POEMS syndrome-associated multicentric Castleman’s disease. Blood 1999;93:3643–3653.
  4. Rubin LJ: Primary pulmonary hypertension. N Engl J Med 1997;336:111–117.
  5. Galie N, Manes A, Uguccioni L, et al: Primary pulmonary hypertension: insights into pathogenesis from epidemiology. Chest 1998;114:184s–194s.
  6. Cool CD, Rai PR, Yeager ME, et al: Expression of human herpesvirus 8 in primary pulmonary hypertension. N Engl J Med 2003;349:1113–1122.
  7. Bull T, Cool CD, Serls AE, et al: Primary pulmonary hypertension, Castleman’s disease and human herpesvirus-8. Eur Respir J 2003;22:403–407.
  8. Montani D, Achouh L, Marsellin AG, et al: Reversibility of pulmonary arterial hypertension in HIV/HHV8 associated Castelman’s disease. Eur Respir J 2005;26:969–972.
  9. Katano H, Ito K, Shibuya K, et al: Lack of human herpesvirus 8 infection in lungs of Japanese patients with primary pulmonary hypertension. J Infect Dis 2005;191:743–745.
  10. Henke-Gendo C, Mengel M, Hoeper M, et al: Absence of Kaposi’s sarcoma-associated herpesvirus in patients with pulmonary arterial hypertension. Am J Respir Crit Care Med 2005;172:1581–1585.
  11. Henke-Gendo C, Schulz TF, Hoeper MM: HHV-8 in pulmonary hypertension. N Engl J Med 2004;350:194–195.
  12. Laney AS, De Marco T, Peters JS, et al: Kaposi sarcoma-associated herpesvirus and primary and secondary pulmonary hypertension. Chest 2005;127:762–767.
  13. Montani D, Marsellin AG, Sitbon O, et al: Human herpes virus 8 in HIV and non-HIV infected patients with pulmonary arterial hypertension in France. AIDS 2005;19:1239–1240.
  14. Nicastri E: Human herpesvirus 8 and pulmonary hypertension. Emerg Infect Dis 2005;11:1480–1482.
  15. Appelbaum L, Yigla M, Bendayan D, et al: Primary pulmonary hypertension in Israel: a national survey. Chest 2001;119:1801–1806.
  16. Davidovici B, Karakis I, Bourboulia D, et al: Seroepidemiology and molecular epidemiology of Kaposi’s sarcoma-associated herpesvirus among Jewish population groups in Israel. J Natl Cancer Inst 2001;93:194–202.
  17. Guttman-Yassky E, Bar-Chana M, Yukelson A, et al: Epidemiology of classic Kaposi’s sarcoma in the Israeli Jewish population between 1960 and 1998. Br J Cancer 2003;89:1657–1660.
  18. Iscovich J, Boffetta P, Franceschi S, et al: Classic Kaposi sarcoma: epidemiology and risk factors. Cancer 2000;88:500–517.