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Department of Pathology, National Institute of Infectious Diseases, and Departments of Pathology and Pediatrics, School of Medicine, Toho University, Tokyo, Japan
Samples of lung tissue, taken at autopsy, from 10 Japanese patients with primary pulmonary hypertension (PPH) and samples of lung tissue from 12 Japanese patients with secondary pulmonary hypertension were tested for the presence of human herpesvirus 8 (HHV-8). All samples from patients with PPH contained plexiform lesions around pulmonary arterial vessels, but immunohistochemistry failed to detect the HHV-8encoded latency-associated nuclear antigen. HHV-8 DNA could not be amplified by polymerase chain reaction for the HHV-8encoded K1 and KS330233 genes in any sample. These data suggest that HHV-8 infection is not associated with PPH in Japanese patients.
Primary pulmonary hypertension (PPH) is a rare disease that leads to severe right heart failure, which is characterized histologically by vascular lesions in the lung and the proliferation of endothelial cells and smooth muscle cells in the pulmonary arterial walls; these conditions then induce luminal obstruction, resulting in elevation of pressure in the pulmonary arteries. Some cases of PPH are associated with genetic mutations in bone morphogenetic protein receptor 2 (BMPR2) [1]. Recently, human herpesvirus 8 (HHV-8)also known as Kaposi sarcoma (KS)associated herpesviruswas identified, by polymerase chain reaction (PCR), in 10 of 16 samples of lung tissue from patients with PPH, and the expression of latency-associated nuclear antigen (LANA), encoded by HHV-8, was detected, by immunohistochemistry, in the vascular "plexiform" lesions in these patients' lungs, suggesting an association between HHV-8 and the pathogenesis of PPH [2]. Because only 2 of these 10 HHV-8positive patients had BMPR2 mutations, HHV-8 infection did not correlate with BMPR2 mutations in these patients [2].
HHV-8 is categorized as a gamma herpesvirus [3], and the seroprevalence of HHV-8 varies geographically. HHV-8 has a high seroprevalence in the general population in African countries (40%) and in southern European countries (10%), but a low prevalence has been suggested in the United States (3%) and in Asian countries, including Japan (1.4%) [4]. HHV-8 has been detected in KS, primary effusion lymphoma (PEL), and some cases of multicentric Castleman disease (MCD) [3]. HHV-8encoded LANA is always expressed in the cells of KS and PEL, suggesting an HHV-8 infection in the latent phase. In contrast, not only LANA but also other lytic antigens of HHV-8 are expressed in the cells of MCD, implying that it has a different pathogenesis than do KS and PEL [5]. LANA, however, plays an important role in the pathogenesis of KS and PEL [3]. The histological features of the plexiform lesions of PPHproliferation of spindle-shaped cells with vascular slitsresemble the histological features of KS [2]. Although mutations of BMPR2 have been detected in some isolated cases of PPH and in some cases of familial PPH in Japan [6], the pathogenesis of most cases of PPH is still unknown. In the present study, we investigated the presence of HHV-8 in the lung tissue from 10 Japanese patients with PPH and from 12 Japanese patients with secondary pulmonary hypertension (SPH).
Subjects, materials, and methods.
During 19812003, 10 Japanese patients with PPH underwent autopsy at Toho University Hospital in Tokyo, Japan, and samples of their lung tissue were taken for analysis; samples of lung tissue were also taken from 12 Japanese patients, living in the Tokyo area, who had SPH and were not infected with HIV (table 1). The mean age of the patients with PPH was 23.4 years (range, 051 years), and the mean age of the patients with SPH was 31.4 years (range, 083 years). Immunohistochemistry was performed to investigate the expression of LANA on cells of lung tissue, as described elsewhere [5]. A rabbit polyclonal antibody to LANA (dilution, 1 : 3000 [5]) and a rat monoclonal antibody to LANA (dilution, 1 : 3000; Advanced Biotechnologies) were used as primary antibodies. Samples of KS tissue obtained from additional patients were used as positive controls. For PCR analysis, DNA was extracted from samples of lung tissue that were fixed in formalin and embedded in paraffin. DNA from a sample of KS tissue obtained from an additional patient was used as a positive control, and DNA from a sample of healthy skin obtained from an additional patient was used as a negative control [5]. PCR was performed, as described elsewhere [7], to detect the KS330233 gene of HHV-8 (HHV-8encoded ORF26). Nested PCR was performed to detect the K1 gene of HHV-8. For the first round of nested PCR, the external primer pair K1SF (forward primer, 5-TTGTGCCCTGGAGTGATT-3) and K1SR (reverse primer, 5-CAGCGTAAAATTATAGTA-3) was used to amplify a 363-bp fragment of the K1 gene of HHV-8 [8]. The conditions for the first round of PCR were 1 cycle at 94°C for 4 min, followed by 35 cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min. For the second round of PCR, the inner primer pair K1VR1F1 (forward primer, 5-TTGCCAATATCCTGGTATTGC-3) and K1VR1R1 (reverse primer, 5-CAAGGTTTGTAAGACAGGTTG-3) was used to amplify a 162-bp fragment of the K1 gene; the same conditions as in the first round of PCR were used. The -globin gene was amplified as a control, as described elsewhere [7].
Results.
To investigate whether HHV-8 was present in the samples of lung tissue from patients with PPH, we first performed immunohistochemistry to detect LANA. Staining with hematoxylin-eosin revealed that all samples from patients with PPH had characteristic plexiform lesions in their pulmonary arteries (figure 1). In samples from patients with PPH, 18124 plexiform lesions were tested (table 1). Some samples from patients with SPH also had plexiform lesions. Immunohistochemistry by use of 2 antibodies to LANA revealed that LANA was not present in any sample obtained from patients with either PPH or SPH (table 1), whereas LANA was detected as a dot-like nuclear staining pattern in samples of KS tissue obtained from control patients (figure 1). Although sclerosing lesions and proliferation of endothelial cells and smooth muscle cells around vessels were observed in the plexiform lesions, LANA was not present. To confirm the results of the immunohistochemistry, we extracted DNA from the samples of lung tissue and performed PCR. Both PCR amplification for the KS330233 gene of HHV-8 and nested PCR amplification for the K1 gene of HHV-8 failed to detect HHV-8 DNA in all samples (table 1). The control gene -globin was detected in all samples. These data and the results of the immunohistochemistry suggest that the patients with PPH did not have HHV-8 infection.
Discussion.
In the present study, we have demonstrated that 10 Japanese patients with PPH and 12 Japanese patients with SPH did not have HHV-8 infection. Although we used testing procedures similar to those employed by Cool et al. [2, 9]immunohistochemistry and PCRour results were completely different from theirs.
Patients with PPH are found worldwide. Only 50% of patients with familial PPH have BMPR2 mutations, and no BMPR2 mutations have been detected in patients with isolated cases of PPH. Because HHV-8 was not detected in 6 of the 16 patients with PPH whom Cool et al. studied, the authors suggested that BMPR2 mutations and HHV-8 infection were not correlated [2]. The present study has demonstrated that all 10 Japanese patients with PPH were negative for HHV-8 infection. Although we were unable to examine the seropositivity of the patients with PPH, in a study published elsewhere, we demonstrated that the seroprevalence of HHV-8 was low (1.4%) in the general population in Japan [4]. These data suggest that PPH might be induced by causative factors other than HHV-8 infection and BMPR2 mutations. Therefore, it is possible that the pathogenesis of PPH in Japan is different from that of PPH in the United States. Other genetic backgrounds, modifier genes, or other pathogens may be associated with cases of PPH in Japan.
The sensitivity and methods used in the present study, however, were different from those used by Cool et al. [2]. Our immunohistochemistry succeeded in detecting LANA in all cases of KS, regardless of the stage of disease or the patient's HIV infection status, and the results of immunohistochemistry correlated well with those of PCR [5]. Cool et al. detected LANA not only in the cells within plexiform lesions but also in bronchoepithelial cells and in inflammatory cells, including lymphocytes and macrophages [2, 9], but we were not able to detect LANA in any cells of the samples obtained from patients with PPH. LANA has been detected only in the nuclei of KS cells and not in surrounding cells, including epithelial cells, lymphocytes, and macrophages, even in samples of lung tissue from patients with KS [5]. To date, HHV-8 has been detected, by PCR, in patients with various diseases, but immunohistochemistry has yielded positive results only in samples from patients with KS, PEL, MCD, and some solid lymphomas [10, 11]. Recently, a low seroprevalence of antibodies to HHV-8 in patients with PPH in Germany was reported, suggesting that HHV-8 infection is rarely involved in the pathogenesis of PPH [12]. Further studies are required to clarify the strict association between HHV-8 infection and PPH.
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