Eosinophilic folliculitis: an important differential diagnosis after allogeneic bone-marrow transplant.

Eosinophilic folliculitis (EF) is a descriptive histopathological term applied to a heterogeneous group of disorders. In EF, the characteristic histopathological features are eosinophilic spongiosis and pustulosis involving the infundibular region of the hair follicle. EF may be seen in association with bacterial and fungal infection, drug reactions and haematological disorders. However, in those conditions, the histopathological changes are rarely restricted to the infundibula but in most cases include a moderate to dense perifollicular or even diffuse dermal infiltrate of lymphocytes, or eosinophilic or neutrophilic granulocytes. We present two cases of EF after mini-allogeneic bone-marrow transplantation (BMT) in order to highlight this rare and perhaps under-recognized clinical association.

The Scotland and Newcastle epidemiological study of Hodgkin's disease: impact of histopathological review and EBV status on incidence estimates.

AIMS: The epidemiological and pathological features of Hodgkin lymphoma (HL) are complex. The Epstein-Barr virus (EBV) is consistently associated with a proportion of cases, and these cases are thought to represent a distinct aetiological subgroup of HL. The aim of the present analysis was to determine the age and sex specific incidence of EBV associated and non-associated HL, analysed separately, using data derived from a population based study-the Scotland and Newcastle epidemiological study of Hodgkin's disease (SNEHD). This study also provided a unique opportunity to evaluate accuracy in the current diagnosis and classification of HL. METHODS: SNEHD analysed consecutive cases of HL diagnosed in the study area between 1993 and 1997. Diagnostic biopsy material was retrieved, EBV status of tumours was determined, and histological review was performed. RESULTS: In total, 622 cases were eligible for the study, and EBV studies and histopathological review were performed on biopsy material from 537 and 549 cases, respectively. Accuracy in the overall diagnosis of HL and classification of nodular sclerosis HL was good, but diagnosis of HL in the elderly and classification of other subtypes was less reliable. One third of classic HL cases were EBV associated, and age specific incidence curves for EBV associated and non-associated cases were distinct. CONCLUSIONS: Comparison of age specific incidence curves for EBV associated and non-associated HL supports the hypothesis that these are two distinct aetiological entities. Accuracy in the diagnosis of HL is generally good, but certain subgroups of cases continue to present diagnostic difficulties.

Epstein-Barr virus infection in colorectal neoplasms associated with inflammatory bowel disease: detection of the virus in lymphomas but not in adenocarcinomas.

Epstein-Barr virus (EBV) is associated with several lymphoid and epithelial human malignancies. The latter include gastric adenocarcinomas, while sporadic colorectal adenocarcinomas (CRCs) have been reported to be EBV-negative. Recently, increased numbers of EBV-infected B lymphocytes have been detected in intestinal mucosal samples affected by ulcerative colitis (UC) and, to a lesser extent, Crohn's disease (CD). Both CRC and colorectal non-Hodgkin's lymphoma (NHL) are recognized complications of inflammatory bowel disease (IBD), but it is unclear to what extent EBV contributes to the development of these neoplasms. Seventeen cases of IBD-associated CRC and nine cases of IBD-associated colorectal NHL were therefore studied for the presence of EBV by in situ hybridization. EBV-positive cases were further studied for the expression of the EBV-encoded nuclear antigen (EBNA) 2 and the latent membrane protein (LMP) 1 of EBV by immunohistochemistry. Four out of seven cases of colorectal NHL associated with UC were shown to be EBV-positive. In addition, two of two colorectal NHLs developing in patients with CD were EBV-positive. Of the EBV-positive lymphomas, three displayed a pattern of EBV latent gene expression consistent with type I latency (EBNA2(-)/LMP1(-)), two a type II pattern (EBNA2(-)/LMP1(+)), and one a type III pattern (EBNA2(+)/LMP1(+)). These findings suggest that EBV infection is involved in the pathogenesis of a proportion of colorectal NHLs developing in IBD. Iatrogenic immunosuppression may contribute to the development of these lymphomas. By contrast, all 17 IBD-associated CRCs were EBV-negative, including a case of CRC occurring synchronously with an EBV-positive NHL. In conjunction with previous reports on sporadic CRCs, this suggests that EBV is not involved in the pathogenesis of CRC.

The ins and outs of EBV infection.

Epstein-Barr virus (EBV) infects almost the entire adult population of the world. The success of this virus appears to be based on its ability to infect the B cell, rather than any other cell type. We review EBV B-cell tropism, and discuss the mechanisms by which the virus may gain access to, and egress from, B cells in the normal host.

Epstein-Barr virus and Hodgkin's disease: further evidence for the three disease hypothesis.

The epidemiology of Hodgkin's disease suggests that it is a heterogeneous condition comprising more than one disease entity. The Epstein-Barr virus (EBV) is present in the Reed-Sternberg cells of a proportion of cases and is likely to play a role in the pathogenesis of these cases. In this study we show that EBV association rates vary with age at diagnosis. We suggest that Hodgkin's disease can be divided into three disease entities on the basis of EBV association and age, thereby providing biological support for the multiple aetiology hypothesis proposed by MacMahon (Cancer Res 1966; 26: 1189-1290).

New monoclonal antibodies to the T cell antigens CD4 and CD8. Production and characterization in formalin-fixed paraffin-embedded tissue.

We have generated a recombinant protein representing part of the CD4 molecule and a peptide representing an epitope of predicted high antigenicity on the CD8 molecule and employed these to generate mouse monoclonal antibodies using standard hybridoma protocols. The extracellular domain of the CD4 molecule was obtained by reverse transcription of mRNA from peripheral blood lymphocytes followed by polymerase chain reaction. The amplified gene fragment was cloned into an expression vector to allow a histidine-tagged fusion protein to be produced in Escherichia coli. Purified fusion protein was used to immunize mice. The CD8 monoclonal antibody was raised against a peptide consisting of 13 amino acids within the carboxyl-terminal region of the CD8 cytoplasmic domain. The antibodies showed appropriate reactivity on Western blotting. By heat pretreatment, these antibodies have been shown to be highly effective on paraffin-embedded tissue. In normal lymphoid tissue, the expected distribution of CD4 and CD8 lymphocytes was observed. In a series of 16 T cell lymphomas and B cell lymphomas, immunostaining results were compared with those obtained using reagents effective only in frozen tissue. A high degree of correlation was observed. These results suggest that NCL-CD4 and NCL-CD8 may be of value in the characterization of T cell disorders.

Determination of HLA-A*02 antigen status in Hodgkin's disease and analysis of an HLA-A*02-restricted epitope of the Epstein-Barr virus LMP-2 protein.

There is good evidence for an association between Epstein-Barr virus (EBV) and Hodgkin's disease (HD). In approximately one-third of cases, the EBV genome is detectable in Reed-Sternberg (RS) cells and there is expression of the viral nuclear antigen EBNA-1 and the latent membrane protein LMP-1. Expression of LMP-2 has been demonstrated at the mRNA level, and it is presumed that the protein is expressed alongside LMP-1. The LMP-2 protein is known to contain an epitope presented to cytotoxic T-cells which is restricted through the HLA class I antigen A*0201 in healthy seropositive individuals. Since most HLA-A*02-positive Caucasians are HLA-A*0201-positive, it was hypothesized that HLA-A*02-positive individuals would be under-represented among Caucasians with EBV-associated HD. HLA-A*02 status was determined, using flow cytometry and/or the polymerase chain reaction, for 276 individuals including 176 cases of HD. There was no significant difference between the frequency of HLA-A*02 positivity in HD cases and controls, and between EBV-associated and non-associated cases of HD. The A*02 alleles of 14 cases of EBV-associated HD were further subtyped using nested PCR; all except one case were found to be A*0201-positive. We therefore investigated whether there was any evidence for mutation of the epitope representing amino acids 426-434 of LMP-2a which is restricted through HLA-A*0201. In 10/11 cases the nucleotide sequence encoding this epitope was identical to the published sequence; in the remaining case there was a mutation which would not be expected to alter the conformation of the epitope. Overall, our data suggest that other mechanisms of immune escape must be operative in EBV-associated HD.

CHOP-based chemotherapy is as effective as alternating PEEC/CHOP chemotherapy in a randomised trial in high-grade non-Hodgkin's lymphoma. Scotland and Newcastle Lymphoma Group.

The aim of this study was to test whether survival for patients with high-grade non-Hodgkin's lymphoma (NHL) can be improved with a non-cross-resistant regimen as compared to a CHOP-based regimen. This is a multicentre study comprising 325 adult patients, median age 58 years, with high-grade non-Hodgkin's lymphoma: patients of any age and performance status were eligible provided they were able to receive the drugs in the regimens. Patients were randomised to either B-CHOP-M (bleomycin, cyclophosphamide, doxorubicin, vincristine, prednisolone and methotrexate) or PEEC-M (methylprednisolone, vindesine, etoposide, chlorambucil and methotrexate) alternating with B-CHOP-M. At a median follow-up of 9 years, there was no significant difference in overall survival or disease-free survival between the two arms. Toxicities for the two regimens were equivalent. This study confirms that for relatively unselected patients with high-grade non-Hodgkin's lymphoma, an alternating multidrug regimen does not improve upon the results obtained with B-CHOP-M.

The immunological architecture of B-lymphocyte aggregates in cryptogenic fibrosing alveolitis.

Cryptogenic fibrosing alveolitis (CFA) is believed to have a pathogenesis mediated by the cellular arm of the immune system. Previous studies have, however, indicated the presence of B-lymphocyte aggregates, as well as evidence of local immunoglobulin production and increased levels of B-cell growth factors. It has recently been shown that CFA is associated with the production of circulating IgG autoantibodies to antigen(s) associated with alveolar lining cells. This prompted an examination of the immunological architecture of the B-lymphocyte aggregates, in order to assess whether they might provide histological confirmation of a local humoral immune response in these patients. Thirty-eight consecutive open lung biopsy specimens were examined from patients with CFA and aggregates of B lymphocytes were identified in 37/38. In only five cases were germinal centres seen. The morphological appearances of the aggregates were reminiscent of those observed in mucosal associated lymphoid tissue (MALT). Using immunohistochemistry, despite the low frequency of true germinal centre formation, the B-lymphocyte aggregates were shown to contain the cellular micro-environment necessary for a humoral immune response. In addition, there was evidence of lymphocyte proliferation and activation within these aggregates. These results provide evidence of a local humoral immune response associated with B-lymphocyte aggregates in the lungs of patients with CFA.

Detection of t(14;18) in British follicular lymphoma using cytogenetics, Southern blotting and the polymerase chain reaction.

Cytogenetics, Southern blotting and PCR were used to detect t(14;18) in 72 British patients with follicular lymphoma. The overall incidence of the translocation was 76%. Cytogenetics was the most successful technique, but 10-30% of translocations detected karyotypically were missed by molecular methods, presumably due to break-points falling outside the range of probes and primers used here. Reliance on molecular detection alone may considerably underestimate the incidence of t(14;18) and it is therefore essential to use the most comprehensive range of probes and primers available.

Primary splenic large cell anaplastic lymphoma associated with HIV infection.

We report a case of primary splenic B-cell CD30 positive large cell anaplastic lymphoma developing in an HIV positive patient. The tumour cells expressed Epstein-Barr virus-associated antigens.

Increased CD45RO expression on T lymphocytes in mediastinal lymph node and pulmonary lesions of patients with pulmonary sarcoidosis.

Sarcoidosis is characterized by a cell-mediated response mediated by the activation of CD4+ T lymphocytes in an environment lacking adequate numbers of regulatory CD8+ T lymphocytes. Immunohistological studies on frozen tissues have shown that sarcoid lesions have activated CD4 helper/inducer T lymphocytes at the centre of granulomata, whereas lymphocytes at the periphery are mainly CD8 suppressor/cytotoxic cells. In this study we investigated the immunohistological distribution of CD45 isoforms of T cells in 29 paraffin-embedded sarcoid lesions in mediastinal and open lung biopsies. Ten of these were assessed quantitatively, with single-staining of serial sections demonstrating a predominance of CD45RO memory T lymphocytes in granulomata and intergranulomatous areas. Ratios of CD45RO:CD45RA T lymphocytes (or the ratio of memory to naive T cells) were 42.0:1 for granulomata and 17.9:1 for intergranulomatous areas of sarcoid lesions counted. This finding is compatible with the hypothesis that nearly all the lymphocytes present in sarcoid lesions have been previously activated, and selectively home to sarcoid lesions.

Epstein-Barr virus in Hodgkin's disease and site of origin of tumour.

Epstein-Barr virus (EBV) may be involved in the pathogenesis of Hodgkin's disease. We investigated whether EBV in Hodgkin's disease is related to the site of origin of the tumour. In 40 patients with stage I disease, there was a significant association between EBV latent membrane protein (LMP-1) expression and presentation in neck lymph nodes. There was no association in stage II-IV disease (57 cases). Nodular sclerosing subtype was rarely associated with LMP-1 expression. In some cases of Hodgkin's disease of mixed cellularity or lymphocyte predominant subtype originating in the neck, EBV may be an important aetiological co-factor.

CD40 expression in Hodgkin's disease.

CD40 is a transmembrane protein that belongs to a superfamily of proteins related to nerve growth factor receptor. CD40 is expressed on B cells and some B cell malignancies. It appears to be involved in B cell proliferation and the prevention of apoptosis in germinal center cells, which is accompanied by expression of bcl-2. Its expression is up-regulated by the EBV protein latent membrane protein-1 and cytokines interleukin-4 and interferon-gamma. The expression of CD40 in 37 cases of Hodgkin's disease and 23 cases of non-Hodgkin's lymphoma (11 T cell lymphomas and 12 B cell lymphomas) was examined by paraffin section immunohistochemistry using the BB-20 monoclonal antibody. In 26 of 37 cases of Hodgkin's disease the Reed-Sternberg cells showed strong membrane or cytoplasmic expression of CD40. Only 3 of 23 non-Hodgkin's lymphomas showed any expression of CD40 and then only weakly. There was no correlation between expression of bcl-2 or latent membrane protein-1 with CD40 expression. These results show that there is probable hyperexpression of CD40 in Hodgkin's disease and suggest that dysregulation of CD40 expression may play a role in the pathogenesis of Hodgkin's disease.

Association of Epstein-Barr virus with pediatric Hodgkin's disease.

A bimodal age incidence curve has been shown for Hodgkin's disease (HD). In developing countries, the first age incidence peak occurs in childhood; however, this peak is delayed until young adulthood in developed countries. This difference may reflect differences in the age of exposure to infectious agents involved in the development of HD or may suggest different etiological agents. Epstein-Barr virus (EBV) has been implicated in the pathogenesis of a proportion of HD cases. In this study, EBV association was investigated in a series of 55 pediatric HD cases from three geographical locations (United Kingdom, Brazil, and Saudi Arabia) and the relationship between country, age, sex, histological subtype, and EBV positivity was evaluated. EBV was detected in 38 cases using RNA in situ hybridization, Southern blot, or immunohistochemical analysis. No significant difference in EBV positivity by country, age, or sex was observed; however, children under 10 years of age were particularly likely to be EBV-associated. The difference in EBV association in the pediatric group compared with that observed previously for young adult HD was highly statistically significant (P < 0.0001). These results are consistent with the hypothesis that pediatric and young adult HD have different etiologies and suggest that EBV is likely to be involved in the pathogenesis of pediatric HD.

Lymphoma in the BB/E rat: c-myc translocation identified.

In a prospective necropsy study involving 257 animals in the BB rat colony in Edinburgh, there was an increased incidence of lymphoma (average 10.9 per cent in all three subgroups: 8 per cent in diabetic, 20 per cent in non-diabetic diabetes-prone, and 3.2 per cent in diabetes-resistant rats). The incidence was significantly increased (P < 0.05) in the non-diabetic diabetes-prone subgroup. These results differ markedly from previous results and indicate that the relationship between lymphoma and diabetes is more complex than previously suggested. All the lymphomas bar one involved the ileocaecal nodes and were classified as immunoblastic lymphomas of B-cell origin. There was a striking resemblance both in tissue distribution and in histological classification to the lymphoma seen in the established B-cell lymphoma model, the LOUVAIN rat. Southern blot analysis carried out on the BB rat lymphomas revealed a translocation of variable length involving the c-myc oncogene. Such a translocation has not been demonstrated in the BB rat before.

B lymphocyte accumulations in human pulmonary sarcoidosis.

BACKGROUND: Although cell mediated immunity is primarily thought to mediate the pathogenesis of sarcoidosis, the presence of immunoglobulins, immune complexes and complement suggests that processes of humoral immunity may contribute to immunopathology in sarcoid lesions. To test this hypothesis, the distribution of B lymphocytes in paraffin embedded sarcoid lesions in mediastinal lymph nodes and open lung biopsy specimens was investigated. METHODS: Paraffin sections from eight open lung and 21 lymph node biopsies from sarcoid patients and five normal and five tuberculous lymph nodes from patients with tuberculosis were stained with a panel of monoclonal antibodies by means of avidin/biotin enhanced immunocytochemistry. RESULTS: Immunohistochemical analysis of the 29 biopsy specimens from the sarcoid patients revealed large numbers of B cells in the intergranulomatous regions. Further investigations in the open lung biopsy specimens indicated that these B cells were often organised into discrete circular or oval shaped aggregates with no germinal centre morphology, in which a few CD45RO memory T lymphocytes were scattered. The B cells were polyclonal, and a few plasma cells (IgM+, IgA+, IgG+) were identified. CONCLUSIONS: The finding of large numbers of B lymphocytes in sarcoid pulmonary lesions is in contrast to bronchoalveolar lavage studies, which have demonstrated proportions of 5% or less of B cells as a total of all immune cells, and therefore indicates that bronchoalveolar lavage may not correctly sample the immune cells of lung interstitial tissue in pulmonary sarcoidosis. The B cells at these sites are the possible origin of some of the humoral changes in the serum and lesions of sarcoid patients. They may also influence the pathogenesis of the disorder by presenting antigen(s) and forming immune complexes at sites of disease activity.

Criteria for the definition of Epstein-Barr virus association in Hodgkin's disease.

There is a clear association between the Epstein-Barr virus (EBV) and Hodgkin's disease (HD). EBV is not, however, detectable within the affected tissues of all cases. The proportion of positive cases varies from 15-79% depending on the assay used to detect EBV. The techniques utilised vary not only in sensitivity but in their ability to detect viral DNA, RNA, or protein and in their ability to demonstrate the cellular localisation of the virus. Thus, the biological significance of a positive result will vary depending on the method of analysis. In the present study, four different methods of detecting EBV were compared. RNA in situ hybridization was found to be the most practical method of detecting EBV in tumour cells. Using this assay EBV was detected in the Reed-Sternberg cells of 33% and 45% of the two series of HD cases examined in this study. We believe that these cases should be considered EBV-associated.

The expression of the EBV latent membrane protein (LMP-1) is independent of CD23 and bcl-2 in Reed-Sternberg cells in Hodgkin's disease.

A series of 33 cases of Hodgkin's disease was investigated for the presence of the EBV encoded latent gene product LMP-1 and of CD23 using immunohistochemical techniques. The expression of bcl-2 was examined in a subset of cases. LMP-1 was detected in the Reed-Sternberg cells in 15 cases. Although LMP-1 is known to upregulate CD23 and bcl-2, there was no correlation between the expression of LMP-1 and the detection of CD23 and bcl-2 in Reed-Sternberg cells.

Lymphocytes in pseudomembranes of late prosthetic joint failure.

The pseudomembrane formed in association with late aseptic prosthesis failure contains a mixed giant cell and histiocytic infiltrate with variable numbers of lymphocytes. Immunolabelling with a panel of antibodies on paraffin sections was undertaken to define the nature of the lymphoid infiltrate in 19 cases. In all cases, the predominant lymphoid cell was a memory (CD45RO+, CD45RA-) T-cell. B-cells were rare. Tissue from patients with rheumatoid arthritis (RA) contained greater numbers of T-cells when compared with patients with osteoarthritis (OA), suggesting that the intensity of the lymphoid infiltrates reflects the underlying joint disease rather than necessarily being part of a hypersensitivity response to wear debris.