High prevalence of Epstein-Barr virus in the Reed-Sternberg cells of HIV-associated Hodgkin's disease.

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease, with an frequency of 15 to 50% in the immunocompetent host. We studied 12 formalin-fixed, paraffin-embedded cases of Hodgkin's disease occurring in human immunodeficiency virus-infected individuals to determine the frequency of EBV in Hodgkin's disease from this population. EBV DNA-RNA in situ hybridization was performed using a 30-base biotinylated anti-sense oligonucleotide complementary to the EBER1 gene of EBV. EBV RNA was found in the Reed-Sternberg cells and variants in 11 of 12 cases. Double-labeling studies confirmed the presence of EBV RNA in CD15-expressing Hodgkin's cells in all 11 cases, although rare B lymphocytes coexpressing EBV RNA and CD20 were also noted in these cases. The Hodgkin's cells in all 11 EBER-positive cases expressed latent membrane protein. The one case negative for EBV RNA showed the histology of nodular, lymphocyte predominance, a subtype thought to be distinct from other types of Hodgkin's disease.     

Hodgkin''s disease, lymphocyte predominance type, nodular--further evidence for a B cell derivation. L & H variants of Reed-Sternberg cells express L26, a pan B cell marker.

Immunoreactivity for L26, a highly effective pan B cell marker that can be detected in paraffin sections, was evaluated in 72 cases of Hodgkin's disease of various histologic types. In all cases of nodular lymphocyte predominance type of Hodgkin's disease, L & H variants of Reed-Sternberg cells uniformly exhibited strong immunoreactivity for L26. Other variants of Reed-Sternberg cells, eg, lacunar, mononuclear, and diagnostic forms, present in nodular sclerosis, mixed cellularity, and lymphocyte depletion types of Hodgkin's disease, infrequently expressed L26 reactivity. In 55 of 63 cases (87%) of these combined types, less than 5% of Reed-Sternberg cells or variants were L26 positive. In the remaining cases, a larger proportion of these cells expressed L26. Topographic patterns of immunoreactivity for small lymphocytes in these different types of Hodgkin's disease also varied. In nodular lymphocyte predominance type, L26 positive lymphocytes (presumptive B cells) were mainly localized to nodular areas of the proliferation. In other types of Hodgkin's disease, L26 positive cells occurred in small or large aggregates and generally represented a minor proportion of the population of lymphoid cells. These studies further support a B cell derivation for L & H variants of Reed-Sternberg cells and provide additional evidence that nodular lymphocyte predominance type Hodgkin's disease may represent a distinct entity, possibly an unusual low grade B cell lymphoma. These data also suggest that some Reed-Sternberg cells and variants present in other histologic types of Hodgkin's disease may be of B cell derivation, and precludes the use of L26 as a diagnostic discriminant in cases in which the distinction between Hodgkin's disease and non-Hodgkin's lymphoma is unclear.     

The nature of the lymphocytes surrounding Reed-Sternberg cells in nodular lymphocyte predominance and in other types of Hodgkin''s disease.

The lymphocytes surrounding Reed-Sternberg cells may play an important role in the pathogenesis of Hodgkin's disease. In this study, T cells in different subtypes of Hodgkin's disease were analyzed in situ by an immunoperoxidase method employing a panel of antibodies, including several paraffin tissue-reactive monoclonal antibodies. The T cells in Hodgkin's disease-involved tissues were found to be activated CD4-positive T cells that are UCHL1+ and CD45R-. This immunophenotype is compatible with an activated helper-inducer memory T cell population. The T cells in the nodular lymphocyte predominance subtype were found to have additional positivity for Leu 7, indicating a subpopulation of CD4+ T cells, normally confined to the light zone of germinal centers of secondary follicles.     

Detection of immunoglobulin light chain mRNA in nodular sclerosing Hodgkin''s disease by in situ hybridization with biotinylated oligonucleotide probes compared with immunohistochemical staining with poly- and monoclonal antibodies

In order to elucidate the origin of the Hodgkin's and Reed-Sternberg cells, the expression of immunoglobulin kappa- and lambda light chain mRNA in 23 cases of nodular sclerosing and two cases of mixed cellularity Hodgkin's disease was examined by in situ hybridization using biotinylated oligonucleotide probes and compared with immunohistochemical staining with mono- and polyclonal antibodies against immunoglobulin kappa- and lambda light chains. No hybridization signals were seen in Hodgkin's or Reed-Sternberg cells in any of the cases. Polyclonal staining with polyclonal anti-immunoglobulin light chain antibodies was seen in Hodgkin's and Reed-Sternberg cells in 12 cases of nodular sclerosis and in two cases of mixed cellularity and with monoclonal antibodies in three cases of nodular sclerosis, but in no cases of mixed cellularity. In all cases, there was polyclonal labelling of plasma cells with both the oligonucleotide probes and the antibodies. In five cases, the Hodgkin's and Reed-Sternberg cells were also stained with one of the B-cell antibodies L26, MB2 or LN1. Lack of mRNA signals in Hodgkin's and Reed-Sternberg cells might indicate that these cells in Hodgkin's disease of the nodular sclerosis subtype are either not B-cell derived or they are early B-cells (precursor B-cells) not yet able to produce immunoglobulin light chain mRNA, at least not at a level detectable by in situ hybridization. Immunohistochemical staining of Hodgkin's and Reed-Sternberg cells, however, with antibodies against immunoglobulin kappa and lambda light chains may be explained by cellular uptake of the light chains, but the difference in reactivity between poly- and monoclonal antibodies cannot be explained at present.     

Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin''s disease is frequently associated with CR2 (EBV receptor) expression

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.     

Immunohistochemical demonstration of the Epstein-Barr virus-encoded latent membrane protein in paraffin sections of Hodgkin's disease.

Paraffin sections from 46 cases of Hodgkin's disease were examined for the presence of the Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP) using a sensitive (double layer alkaline phosphatase-anti-alkaline phosphatase) immunohistochemical method. LMP was detected in 22 cases, the majority of positive cases being of nodular sclerosis (12/24), mixed cellularity (6/7), and lymphocyte depletion (3/3) subtypes. Only one of 12 cases of lymphocyte predominant disease was positive. In all cases, reactivity was confined to Hodgkin's and Reed-Sternberg cells. These results provide further evidence for an association between EBV and Hodgkin's disease and indicate that LMP may be readily detected in archival material.     

Demonstration of Epstein-Barr virus genome in neoplastic cells of Hodgkin's disease by in situ hybridization, in paraffin-embedded tissue using biotinylated probes. A study of 46 cases.

Paraffin-embedded tissue specimens from 46 cases of Hodgkin's disease (HD) were studied by in situ hybridization with biotinylated probes for the presence of EBV genomes. EBV specific DNA sequences were detected in the nuclei of Reed-Sternberg (RS) and Hodgkin cells (H), in 14 of these 46 cases. There was no correlation between positive hybridization and morphological subtype or site of tumor. By demonstrating an exclusive localization of the viral DNA in the tumor cells of HD, our study adds to the growing body of evidence to suggest an involvement of EBV in the pathogenesis of at least some cases of HD.     

Paragranuloma is a variant of Hodgkin's disease with predominance of B-cells

Summary Fifteen cases of Hodgkin's disease of nodular and diffuse paragranuloma subtype (nodular and diffuse subtype of lymphocyte predominant type of Hodgkin's disease) were studied by light and electron microscopy, using monoclonal antibodies recognizing T and B-lymphocytes and dendritic reticulum cells. The results were compared with findings on 10 cases of Hodgkin's disease of mixed type with lymphocyte predominance. The present study provides evidence that paragranuloma represents a special variant of Hodgkin's disease different from other subtypes. Paragranuloma is characterized by predominance of B-cells which were demonstrated with a new B cell reagent KiB3 on routinely processed paraffin sections.This paper is dedicated to Professor Karl Lennert, Kiel, on the occasion of his 65th birthday     

Epstein-Barr virus is associated with all histological subtypes of Hodgkin lymphoma in Vietnamese children with special emphasis on the entity of lymphocyte predominance subtype

Early acquisition of Epstein-Barr virus (EBV) infection is prevalent in developing countries. We studied infectious mononucleosis (IM) and the subtypes of Hodgkin lymphoma (HL) with the status of EBV infection in Vietnamese children. Among the 46 cases of HL, the male-to-female ratio was 38:8, and the mean age at presentation was 6.6 years. Similar to the subtype distribution in developed countries, cases were classified as nodular sclerosis (NSHL) subtype in 56.5% (n = 26), mixed cellularity (MCHL) in 23.9% (n = 11), lymphocyte-rich classic (LRCHL) in 8.7% (n = 4), lymphocyte depletion (LDHL) subtype in 4.4% (n = 2), and nodular lymphocyte predominance (NLPHL) subtype in 6.5% (n = 3). However, in situ hybridization for EBV-encoded RNA revealed that the tumor cells were positive in 93.2% (41/44) of cases, including all 3 cases of nodular lymphocyte predominance HL. Expression of CD20 on Reed-Sternberg cells could be demonstrated in 17% (7/42) of classic HL. The high incidence of EBV in these cases of HL was correlated with an earlier mean age of presentation of primary EBV infection (ie, IM), at 5.3 years, in this patient population, compared with an average of 15 to 19 years reported in developed countries. This study demonstrates that in an area with an earlier mean age of onset of EBV infection, nearly all cases of pediatric HL, including all histological patterns, may be related to EBV infection. The association of NLPHL with EBV is unusual, and the literature is reviewed and discussed.     

Hodgkin's disease,lymphocyte predominance type,nodular--a distinct entity? Unique staining profile for L&H variants of Reed-Sternberg cells defined by monoclonal antibodies to leukocyte common antigen,granulocyte-specific antigen,and B-cell-specific antigen.

Using monoclonal antibodies to leukocyte common antigen, granulocyte-related antigen, and B-cell specific antigens, L&H variants of Reed-Sternberg (R-S) cells in Hodgkin's disease, lymphocyte predominance type (nodular), exhibited a unique staining profile as compared with R-S cells of other histologic types. L&H variants were strongly immunoreactive for leukocyte common antigen, as defined by monoclonal antibodies PD6/27 and 2B11; whereas other types of R-S cells were negative or rarely positive. R-S cells and variants in 69 cases of Hodgkin's disease of nodular sclerosis (41), mixed cellularity (25) or lymphocyte depletion (3) types, were consistently strongly immunoreactive for Leu-M1, a granulocyte-related antigen, while L&H variants were uniformly nonreactive (4 cases). B-cell specific antigens, detected by three pan-B-cell monoclonal antibodies, were observed only for L&H variants. These observations suggest that L&H variants of R-S cells represent a distinct type of transformed cell, possibly of B-cell origin, and do not share a common lineage with other types of R-S cells. These studies provide further evidence that Hodgkin's disease, lymphocyte predominance type, nodular, may represent a distinct entity.     

Co-expression of Epstein-Barr virus latent membrane protein and vimentin in “aggressive” histological subtypes of Hodgkin's disease

Summary The presence of Epstein-Barr virus (EBV) genome in Hodgkin's and Reed-Sternberg (HRS) cells, as detected using in situ hybridization (ISH) with biotinylatedBamHI V probes, along with the expression of EBV-encoded latent membrane protein (LMP) and vimentin was examined in paraffin-embedded sections of 39 immunomorphologically characterized cases of Hodgkin's disease (HD). ISH demonstrated EBV in HRS cells in 15 of 39 cases, whereas LMP expression was detected in 11 of 39 cases, only in the presence of EBV genome detection. With the exception of 1 case, in which HRS cells expressed B-cell-associated antigens, the LMP-positive cases included specimens in which HRS cells were of non-B, non-T phenotype. LMP expression showed a stronger association with lymphocyte depletion (LD) (3/3) and mixed cellularity (MC) (6/11) than with lymphocyte predominance (0/5) or nodular sclerosis (2/20) subtypes. Vimentin expression on HRS cells was found in all the LMP-expressing cases and only in a fraction (13/28) of LMP-negative cases. This study supports the view that HD represents a heterogeneous group of diseases also in terms of EBV association, LMP expression being strongly related to the aggressive LD and MC histological subtypes. In light of the supposed interactions between vimentin and LMP, their coexpression on HRS cells, as detected in this study, provides further evidence for a significant role of EBV in the development of a proportion of HD cases.     

Epstein–Barr virus (EBV) and Hodgkin's disease in a multi-ethnic population in Malaysia

The Epstein–Barr virus (EBV) has been implicated as a contributing factor in the development of Hodgkin's disease. Western cases of Hodgkin's disease have shown the presence of EBV in Hodgkin and Reed–Sternberg cells in approximately 50%. We studied a total of 100 consecutive cases of Hodgkin's disease from Malaysia, with the aim to elucidate its association with EBV in a multi-ethnic Asian population. Of 34 patients (34%) less than 15 years of age (childhood), 25 had classical Hodgkin's disease (eight nodular sclerosis, 16 mixed cellularity, one lymphocyte depleted) and nine had lymphocyte predominance Hodgkin's disease. Of the 66 from patients aged 15 years and above, 33 had nodular sclerosis, 24 mixed cellularity, two lymphocyte depleted, one unclassifiable and six lymphocyte predominance Hodgkin's disease. The ethnic distribution of classical Hodgkin's disease was: Malay 23, Chinese 32 and Indian 30 (Malay:Chinese:Indian = 1:1.4:1.3), and the ethnic distribution in the 15 cases of lymphocyte predominance Hodgkin's disease was: Malay four, Chinese 10 and Indian one. Taking into account the ethnic distribution of the general population and of hospital admissions, there appears to be a significant predilection of classical Hodgkin's disease cases in ethnic Indian compared to non-Indian patients (chi-squared test, 0.025 > P > 0.01). Eighty-one cases were tested for the presence of EBV by in situ hybridization for EBV encoded RNA, and 57 cases by immunostaining for EBV latent membrane protein 1. In the younger age group, all except one of the 15 cases (nine mixed cellularity, six nodular sclerosis) showed the presence of EBV (93%). In the older age group, EBV was detected (52%) in the following proportion: 6/27 nodular sclerosis, 19/22 mixed cellularity, 1/2 lymphocyte depleted, 1/1 unclassifiable. None of the 14 cases of lymphocyte predominance Hodgkin's disease showed the presence of EBV in the Hodgkin and Reed–Sternberg cells. The findings suggest a strong association of EBV with Hodgkin's disease in Malaysians (41/67, 61%), in particular childhood cases (93%). In adults, the association with EBV is significantly higher in the mixed cellularity subtype (86%) compared with the nodular sclerosis subtype (22%).     

Lymphocyte predominance Hodgkin''s disease—an immunohistochemical study

Lymph node biopsies from 57 local and referred cases, previously diagnosed at Southampton between 1978 and 1987 as lymphocyte predominance Hodgkin's disease were examined using the monoclonal antibodies MT1, UCHL1, L26, LN-1, E29/68 (EMA), Leu-M1 (CD15) and Ber-H2 (CD30). Of the 34 cases with a nodular architecture, 21 (19 male, two female) contained polylobated Reed-Sternberg cell variants with a B-cell phenotype, which lacked expression of CD15. In all cases, the polylobated cells showed positive staining with L26 and LN-1. Six cases expressed EMA and three showed positive staining with Ber-H2. Two cases lacking polylobated cells were reclassified as reactive follicular hyperplasia with progressive transformation of germinal centres. The remaining 11 cases had an atypical immunophenotype and were reclassified, mainly as mixed cellularity Hodgkin's disease. In six cases, the lymph node architecture showed a mixture of nodular and diffuse growth patterns. Five of these cases contained polylobated cells with the typical morphology and immunophenotype of those seen in nodular lymphocyte predominance Hodgkin's disease. The sixth case contained cells expressing CD15, and was reclassified as nodular sclerosing Hodgkin's disease. Of the fifteen biopsies with a diffuse architecture, four contained polylobated B-cells lacking expression of CD15. These were considered to be diffuse lymphocyte predominance Hodgkin's disease. The remaining 11 cases were reclassified as either Hodgkin's disease, mixed cellularity or as T-cell lymphomas.     

Combination of Hodgkin's disease and diffuse large cell lymphoma: an in situ hybridization study for immunoglobulin light chain messenger RNA

It is not clear whether the rare combination of Hodgkin's disease with non-Hodgkin lymphomas are true composite lymphomas or differentiation stages of one tumour cell clone. We used in situ hybridization and immunohistochemistry for the demonstration of immunoglobulin light chains in order to investigate the relationship between the two lymphoma components. In three cases of nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma the Hodgkin cells, as well as the tumour cells in the diffuse large B-cell lymphoma, showed the same messenger RNA for one light chain. Thus, using in situ hybridization in nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma in a small number of cases a possible genetic relationship between the two components could be shown. In nodular sclerosis combined with diffuse large B-cell lymphoma, in situ hybridization did not support a common clonal origin of both tumour parts. However, a unique clonal derivation cannot be excluded by the techniques applied.     

An immunohistological study of the cellular constituents of Hodgkin''s disease using a monoclonal antibody panel

Cryostat sections of lymphoid tissue from 44 cases of Hodgkin's disease were analysed by immunoperoxidase staining using a panel of monoclonal antibodies which included reagents reactive with T cells and their subsets, B cells, HLA-DR, Ig, dendritic reticulum cells and C3b receptor. A wide spectrum of immunohistological patterns was observed ranging from cases in which T cells were numerous (B cells being absent or present in only small numbers) to cases in which very prominent B cell follicles were present. These follicles contained a meshwork of dendritic reticulum cells and were composed of polyclonal B cells (as assessed by light chain expression). T cells were present in small numbers within these B cell follicles, often clustered in a thin rim around individual Reed-Sternberg and Hodgkin's cells. All B cell-rich cases were examples of lymphocyte predominant Hodgkin's disease. Assessment of the T cell helper/suppressor ratios was hindered by the fact that both anti-helper antibodies (OKT4 and anti-Leu 3a) reacted with macrophages. However the majority of cases appeared to contain a normal excess of T helper cells. HLA-DR was strongly expressed in T cell rich areas, on Reed-Sternberg and Hodgkin's cells, on vascular endothelium and on numerous infiltrating cells in the fibrous tissue areas in cases of nodular sclerosing disease. Reed-Sternberg and Hodgkin's cells were not labelled by either anti-fibronectin or by antibodies reactive with dendritic reticulum cells (anti-C3b receptor and antibody R4/23).     

Large B-cell lymphoma rich in T-cells and simulating Hodgkin''s disease

Unusual clinicopathological features drew our attention to nine of 208 cases diagnosed as Hodgkin's disease. Lymph node biopsy specimens in these cases were immunostained with monoclonal antibodies against B-cell, T-cell and Reed-Sternberg cell associated antigens and epithelial membrane antigen (EMA). Reed-Sternberg-like and other atypical large cells were dispersed in a diffuse, small lymphocyte-rich background, consistent more often with the initial diagnosis of diffuse, lymphocyte predominance Hodgkin's disease. The clinical stage in these cases was unusually advanced (stages III and IV). Splenomegaly was a common feature (six of nine cases), the male to female ratio was 7:2 and the median age was 55 years (range 25-77). Response to recognized regimes for Hodgkin's disease treatment was poor in most cases, and three patients died early of their disease. Large cells were B-lymphocytes expressing EMA--an immunophenotype similar to nodular, lymphocyte predominance Hodgkin's disease. Reed-Sternberg cell and T-cell associated antigens were absent on large cells. Mature T-cells, with nuclear irregularities in some instances, predominated in the background. A more appropriate diagnostic category is, therefore, T-cell-rich B-cell lymphoma. The cases represent a 4-5% erroneous diagnosis of Hodgkin's disease and further suggest that there is a need for revision of criteria for the diagnosis of the diffuse, lymphocyte predominance variant.     

Plasmacytoid monocytes (so-called plasmacytoid T cells) in Hodgkin's disease

The occurrence and distribution of plasmacytoid monocytes (so-called plasmacytoid T cells) were investigated immunohistochemically in 40 cases of Hodgkin's disease. Large numbers of plasmacytoid monocytes were found in all cases of lymphocyte predominance, nodular sclerosing, and mixed cellularity Hodgkin's disease, characterized by a minor degree of architectural effacement. They occurred at the periphery of lymphoid aggregates which mimic the composite nodule of the reactive lymph node and which contained Reed-Sternberg cells and their variants. Despite some immunophenotypic similarities, no further arguments were found to support a relationship between plasmacytoid monocytes and Reed-Sternberg cells. We conclude that plasmacytoid monocytes represent one of the monocyte-derived cells that contribute to the cellular reaction in Hodgkin's disease.     

Expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells independent of presence of Epstein-Barr virus.

AIMS: To evaluate the expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells in Hodgkin's disease, especially in relation to Epstein-Barr virus infection and expression of EBV encoded latent membrane protein (LMP). METHODS: Tissues from 33 cases of Hodgkin's disease were studied for the presence of EBV DNA by polymerase chain reaction (PCR) and DNA in situ hybridisation (DISH), for the presence of EBER-1 and EBER-2 EBV RNA by RNA in situ hybridisation (RISH); and for the presence of LMP, bcl-2, and c-myc proteins by immunohistochemical staining. RESULTS: A substantial number of Reed-Sternberg cells expressed bcl-2 in 20 of 29 (69%) and c-myc in 30 of 32 (94%) Hodgkin's disease samples. In 18 of the 25 (72%) cases Reed-Sternberg cells expressed both oncogene products. Of these 18 cases, 10 (56%) were EBV-PCR positive; eight (44%) were EBV-PCR negative. CONCLUSIONS: Reed-Sternberg cells in Hodgkin's disease frequently express both bcl-2 and c-myc oncogene products, suggesting that these oncogenes may act in concert in the pathogenesis of the disease. Moreover, the expression of c-myc and bcl-2 proteins in Reed-Sternberg cells is independent of EBV and LMP status.     

Epstein-Barr virus (EBV) and Hodgkin's disease in children: incidence of EBV latent membrane protein in malignant cells.

Previous studies have detected EBV DNA by Southern blotting or in situ hybridization in biopsy material from up to 30 per cent of adult cases of Hodgkin's disease. Here we have used monoclonal antibodies specific for the EBV latent membrane protein LMP1 to examine archival material from children with Hodgkin's disease. Material from 74 cases (54 males and 20 females) was examined and 37 (30 males and 7 females) were classified as LMP1-positive in the malignant cells. LMP1 positivity was present in 4/13 (31 per cent) of lymphocyte predominant, 14/36 (39 per cent) of nodular sclerosis, 17/20 (85 per cent) of mixed cellularity, 1/2 (50 per cent) of lymphocyte depletion, and 1/3 (33 per cent) of unclassified subtypes. The positive cases by clinical stage were I 9/22 (41 per cent), II 9/20 (45 per cent), III 11/24 (46 per cent), and IV 8/8 (100 per cent). LMP1 positivity was present in 2/5 (40 per cent) children aged less than 5 years, 12/27 (44 per cent) aged 5-10 years, and 23/42 (48 per cent) aged between 10 and 15 years. The association between EBV and Hodgkin's disease in children thus appeared to be more frequent in patients with mixed cellularity and advanced disease, but examples of EBV-positive tumours were found in all histological subtypes, stages, and ages. Stepwise discriminant function analysis showed that clinical stage IV and mixed cellularity histology are independently associated with LMP1 positivity. These observations indicate that Hodgkin's disease in children is at least as strongly linked to EBV as is the disease in adults.