Isolation of viable Hodgkin and Reed-Sternberg cells from Hodgkin disease tissues

Hodgkin disease (HD) is characterized by a small number of malignant Hodgkin and Reed-Sternberg (H/RS) cells among a major population of nonmalignant cells. The analysis of H/RS cells has been hampered by their low frequency and fragility. Here, we describe the isolation of viable H/RS cells from HD affected tissues by high gradient magnetic cell sorting (MACS) according to expression of CD30. The cells were enriched to a purity of up to 50%. H/RS cells were distinguished from other CD30(+) cells by the expression of CD15, their size and granularity. No CD30/CD15 double-positive cells could be enriched from a lymph node affected by the lymphocyte predominant subtype of HD, activated lymph nodes or peripheral blood of healthy donors. For two cases of HD individual MACS-purified H/RS cells and H/RS cells micromanipulated from tissue sections of the same lymphoma specimens were analyzed for Ig gene rearrangements. In both cases, identical V gene rearrangements were amplified from both sources of H/RS cells, showing that H/RS cells were successfully enriched. Moreover, the finding that in both cases no additional Ig gene rearrangements other than the ones identified in the H/RS cells micromanipulated from tissue sections were amplified from the MACS-purified H/RS cells further supports the monoclonality of these cells throughout the affected lymph nodes. The isolation of viable H/RS cells ex vivo is prerequisite for a direct study of gene expression by those cells and of their interaction with cells in their vicinity.     

Origin of the Hodgkin/Reed-Sternberg cells in chronic lymphocytic leukemia with "Hodgkin's transformation"

A lymphoma with the characteristic features of Hodgkin's disease (HD) occasionally develops in patients with B-cell chronic lymphocytic leukemia (CLL), and has been called Richter's syndrome with HD features. In such cases, large tumor cells have the morphological and immunophenotypic features of classical Hodgkin and Reed-Sternberg (H-RS) cells. However, it is not known whether the H-RS cells arise from transformation of the underlying CLL cells or from a different pathological process. We report herein a study of the clonal relationship between the CLL cells and the H-RS cells in three cases of Richter's syndrome with HD features by using a single cell assay. We isolated single CLL cells and H-RS cells from immunostained tissue sections by micromanipulation. The immunoglobulin heavy chain gene (IgH) complementarity determining region (CDR) III of each cell was amplified by the polymerase chain reaction (PCR). The products were then compared by gel electrophoresis and nucleotide sequencing. The IgH CDRIII sequences from the H-RS cells were identical to those from the CLL cells in two cases. In one case, the clonal relationship between the two types of cells could not be determined because PCR products could not be obtained from any of the H-RS cells. This study shows that the H-RS cells and the CLL cells belong to the same clonal population in some cases of Richter's syndrome with HD features. Furthermore, our findings indicate that mature B cells can undergo transformation to cells with the features of H-RS cells, in association with a cellular background typical of HD. This study also supports recent findings suggesting that the H-RS cells in classical HD are derived from transformed B cells.     

Detection of clonal Hodgkin and Reed-Sternberg cells with identical somatically mutated and rearranged VH genes in different biopsies in relapsed Hodgkin's disease

Hodgkin's disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg (H-RS) cells are rare and surrounded by abundant reactive cells. Single-cell analyses showed that H-RS cells regularly bear clonal Ig gene rearrangements. However, there is little information on the clinical evolution of HD in a given patient. In this study, we used the single-cell polymerase chain reaction (PCR) to identify H-RS cells with clonal Ig gene rearrangements in biopsy specimens of patients with relapsed HD. The obtained clonal variable region heavy-chain (VH) gene rearrangements were used to construct tumor-clone-specific oligonucleotides spanning the complementarity determining region (CDR) III and somatically mutated areas in the rearranged VH gene. A number of biopsies were obtained during a period of 3 years from two HD patients. H-RS cells with identical VH rearrangements were detected in two separate infiltrated lymph nodes from one patient with nodular sclerosis HD. In a second patient with mixed cellularity HD subtype, clonal VH rearrangements with identical sequences were detected in infiltrated spleen and two lymph node biopsies. Despite the high sensitivity of the PCR method used (one clonal cell in 10(5) mononuclear cells), residual H-RS cells were not found in peripheral blood, leukapheresis material, purified CD34(+) stem cells or bone marrow. The results show that different specimens from relapsed patients suffering from classical HD carry the same clonotypic IgH rearrangements with identical somatic mutations, demonstrating the persistence and the dissemination of a clonal tumor cell population. Thus, PCR assays with CDRIII-specific probes derived from clonal H-RS cells are of clinical importance in monitoring the dissemination of HD and tumor progression and could be useful for analysis of minimal residual disease after autologous stem cell transplantation.     

Hodgkin and Reed-Sternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal center-derived tumor B cells

Among the four subtypes of Hodgkin disease (HD), lymphocyte-predominant (LP) HD is now generally considered as a separate entity. The B cell nature of the typical Hodgkin and Reed-Sternberg (HRS) cells and their variants (L and H, lymphocytic and histiocytic cells) in LP HD has long been suspected, but the question of whether these cells represent a true tumor clone is unclear. We previously demonstrated clonal Ig gene rearrangements in one case of LP HD. In the present study, five cases of LP HD were analyzed by micromanipulation of single HRS cells from frozen tissue sections and DNA amplification of rearranged Ig heavy chain genes from those cells. Clonal V gene rearrangements harboring somatic mutations were detected in each case. In three cases ongoing somatic mutation was evident. This shows that HRS cells in LP HD are a clonal tumor population derived from germinal center B cells. The pattern of somatic mutation indicates that HRS cells in LP HD are selected for antibody expression. This, and the presence of ongoing mutation discriminates LP from classical HD.     

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined.     

Bcl-2 oncogene protein is preferentially expressed in Reed-Sternberg cells in Hodgkin's disease of the nodular sclerosis subtype

One hundred three cases of nodular sclerosis (NS) and mixed-cellularity Hodgkin's disease were evaluated for expression of bcl-2 oncogene protein, because previous studies have revealed expression of bcl-2 in these subtypes but only rarely in the nodular lymphocyte-predominance subtype. Reed-Sternberg (RS) cells and lacunar variants were positive for bcl-2 in 51 of 86 NS cases and 4 of 17 mixed-cellularity cases. In individual cases of NS, the percentage of RS cells and lacunar variants positive for bcl-2 ranged from minimal (in 5 cases) to 100% positive (mean, 34%). By univariate analysis, expression of the bcl-2 gene product in RS cells was observed in a significantly greater proportion of NS Hodgkin's disease cases than MC cases (P < .009), a finding that may have implications on the pathogenesis of this disorder.     

The B7/BB1 antigen is expressed by Reed-Sternberg cells of Hodgkin's disease and contributes to the stimulating capacity of Hodgkin's disease-derived cell lines

The B7/BB1 molecule has recently been found to be expressed on professional antigen-presenting cells and to be the natural ligand for CD28 and CTLA-4 on T cells. On binding of B7/BB1, CD28 transduces a signal that synergizes with triggering of the T-cell antigen receptor, resulting in enhanced cytokine secretion. In view of the data supporting an antigen-presenting function of Reed-Sternberg cells, we evaluated the expression of B7/BB1 in lymph nodes affected by Hodgkin's disease. B7/BB1 was found to be strongly expressed by the Reed-Sternberg cells in all 47 cases of Hodgkin's disease studied. Moreover, Reed-Sternberg cells were frequently surrounded by CD28-expressing T cells. Evidence for a functional role of B7/BB1 on Reed-Sternberg cells was obtained by our findings that T-cell proliferation and interleukin-2 (IL-2) production in the primary allogenic mixed lymphocyte reaction (MLR), using the B7/BB1-expressing Hodgkin's disease-derived cell lines L428 and KM-H2 as stimulators, could be partially blocked by adding anti-B7 monoclonal antibody. B7/BB1 expression was also evaluated in a group of non-Hodgkin's lymphomas (n = 46). Whereas B7/BB1 was not expressed by the neoplastic cells of most non-Hodgkin's lymphomas, including T-cell-rich B-cell lymphoma (n = 11), it was present on the neoplastic cells of anaplastic large-cell lymphoma (Ki-1 lymphoma) (n = 5) and follicular lymphoma (n = 4). Our data provide further evidence for an accessory cell function of Reed-Sternberg cells. The accessory cell function of Reed-Sternberg cells might lead to pronounced T-cell activation in vivo, which might contribute to the Hodgkin's syndrome. In addition, our study indicates that B7/BB1 may be a useful marker for differentiating Hodgkin's disease from morphologically similar conditions such as T-cell-rich B-cell lymphoma.     

Generation of cells with Hodgkin's and Reed-Sternberg phenotype through downregulation of CD99 (Mic2)

Despite the fact that Hodgkin's and Reed-Sternberg (H-RS) cells are morphological hallmarks of Hodgkin's disease (HD), the nature of H-RS cells still remains to be resolved. Here we report that downregulation of CD99 (Mic2) leads to the generation of cells with an H-RS phenotype. IM9 and BJAB B-cell lines that were transfected with an antisense CD99 expression construct showed the morphological and immunological characteristics of H-RS cells such as multinuclearity, expression of CD15, decreased expression of major histocompatibility complex (MHC) class I and CD45RB, and deregulated secretion of cytokines. The reduced expression of CD99 was also confirmed in H-RS cells of patient's lymph nodes and three HD-derived cell lines, L428, KM-H2, and HDLM-2. Moreover, features characteristic of H-RS cells were completely abolished by forced expression of CD99 and by a constitutively active form of Rac, which functions downstream of CD99. We suggest that CD99 molecules play a crucial role in regulating functions and morphology of cells through a Rac-Rho signaling pathway and that the loss of CD99 expression is a significant molecular event to generate H-RS cells.     

Characteristics and responses of EBV immortalized B cells from periodontal disease patients

OBJECTIVE: This study was designed to examine human B cell responses to Actinobacillus actinomycetemcomitans (Aa). The general hypothesis to be tested was that Epstein-Barr virus (EBV) immortalized B cells could be used to investigate variations in B cell responsiveness of periodontitis patients to periodontal pathogens, and that B cells derived from the peripheral blood of periodontal disease patients infected with Aa demonstrate differences in in vitro activities compared to periodontally healthy subjects. DESIGN: EBV-transformed B cell lines were used to analyze immunoglobulin and Aa-specific antibody responses, as well as to determine the frequencies of cells producing immunoglobulin (Ig) of a specific isotype and detect clones secreting antibodies specific for Aa. Lymphoblastoid cells lines (LCL) were derived by clonal transformation of peripheral blood lymphocytes from 10 Aa-infected patients with adult periodontitis (Aa-AP) and seven normal subjects. METHODS: The B cells were incubated in Aa-coated polystyrene plates to separate adherent and non-adherent cells, and stimulate the cells with the whole bacteria. In addition, the B cells were stimulated with Aa LPS, E. coli LPS, or the polyclonal B cell activators (PBAs), pokeweed mitogen (PWM) and Staphylococcus aureus protein A (SpA). Both adherent and non-adherent cell populations were cultured for up to 15 days. MAIN OUTCOME MEASURE: Total immunoglobulins (Igs) and antibody (IgG, IgA, IgM) levels to Aa in the culture supernatants were assessed using an ELISA. The distribution of IgG, IgA, IgM and Aa-specific antibody producing cells was analyzed by a double immunoenzymatic staining technique. RESULTS: IgM levels produced by the LCLs were significantly increased vs IgG and IgA (P < 0.001). Three days after Aa stimulation, a marked increase in the level of total Igs and Aa-specific antibody was observed in adherent cells from Aa-AP (P < 0.05-0.03). Aa-specific antibody levels were significantly higher in the supernatants from Aa-AP vs normals throughout the culture interval (P < 0.03). There was also a significant increase in Aa-specific antibody levels after stimulation with Aa LPS or E. coli LPS (P < 0.05), whereas PWM and SpA had no significant effect on antibody to Aa. There was a predominance of IgM cells compared to IgG and IgA isotypes (P < 0.04) in LCLs from Aa-infected patients. After stimulation with Aa, a significant increase in the number of IgA (111%) and IgG (48%) secreting cells was observed, concomitant with a 74% decrease in the Ig-negative cell population. Total Aa+ cells increased significantly after stimulation (P < 0.001), predominated by Aa-specific IgG and IgM antibody producing cells. CONCLUSIONS: These results showed that LCLs from Aa-infected patients were polyclonal with respect to isotype distribution. Further stimulation with Aa revealed a shift to cytoplasmic IgG and IgA expression, as well as increases in the Aa-specific B cell population. In contrast, the PBAs stimulated the LCLs to synthesize primarily IgM. Additionally, the findings indicated that: (1) without T cells, polyclonal activation of B cells may lead to elevated Aa-specific B cell populations; and (2) the presence of previously sensitized B cells is required to exert an antigen specific antibody response in the LCL. We conclude that secondary activation of primed B cells by oral bacteria or their products in advanced periodontal lesions may contribute to the local accumulation of significant numbers of Ig-producing cells. This report also suggested that EBV-mediated transformation can be used to probe B cell-bacterial interactions in studies of periodontitis.     

Lack of correlation between the detection of Chlamydia trachomatis DNA in synovial fluid from patients with a range of rheumatic diseases and the presence of an antichlamydial immune response

OBJECTIVE: To resolve how frequently Chlamydia trachomatis and Chlamydia pneumoniae DNA are present in the joints of unselected patients with reactive arthritis (ReA) and undifferentiated oligoarthritis, and to determine if there is an accompanying serologic or cellular antichlamydial immune response. METHODS: Two polymerase chain reaction (PCR) protocols to detect the plasmid of C. trachomatis and the outer membrane protein 1 gene of C. pneumoniae were developed for specific use with synovial fluid (SF). Subsequently, the assays were used to detect DNA from the 2 organisms in SF from 54 adult patients with rheumatic diseases, including 4 with sexually acquired ReA and 31 with undifferentiated oligoarthritis. The presence of chlamydial antibodies and SF lymphocyte proliferation responses were determined in parallel. RESULTS: The PCR protocols were species-specific and highly sensitive. SF samples from 15 patients (8 with undifferentiated oligoarthritis, 3 with ReA, 1 with rheumatoid arthritis, and 1 with psoriatic arthritis) were positive for C. trachomatis. There was no significant correlation between the presence of C. trachomatis DNA in the joint and a Chlamydia-specific synovial T cell response or a serologic response. C. pneumoniae was not detected in any of the 54 patients, although it was identified in the SF from a rheumatoid arthritis patient outside this study, demonstrating that the assay was capable of detecting the organism in the joint. CONCLUSION: C. trachomatis DNA was present in ReA patients and in nearly one-third of unselected patients with undifferentiated oligoarthritis, which further supports the hypothesis that it plays an important role in disease pathogenesis. However, its presence did not correlate with evidence of an antichlamydial immune response. Despite previous anecdotal reports, C. pneumoniae does not appear to be a major cause of undifferentiated oligoarthritis or ReA.     

Long-term depletion of naive T cells in patients treated for Hodgkin's disease

We investigated the representation of T cells in patients who had been treated for Hodgkin's disease (HD). We found a marked depletion in both CD4 and CD8 naive T-cell counts that persists up to 30 years after completion of treatment. In contrast, CD4 and CD8 memory T-cell subsets recovered to normal or above normal levels by 5 years posttreatment. Thus, the previously-reported long-term deficit in total CD4 T-cell counts after treatment for HD is due to specific depletion of naive T cells. Similarly, total CD8 T-cell counts return to normal by 5 years only because CD8 memory T cells expand to higher than normal levels. These findings suggest that the treatment (mediastinal irradiation) results in a longterm dysregulation of T-cell subset homeostasis. The profound depletion of naive T cells may explain the altered T-cell function in treated patients, including the poor response to immunization after treatment for HD. Further, in some individuals, we identified expansions of unusual subsets expressing low levels of CD8. Eight-color fluorescence-activated cell sorting analyses showed that these cells largely express CD8alphaalpha homodimers and CD57, consistent with the phenotype of potentially extrathymically derived T cells. In addition, these cells, both CD4+ and CD4-, are probably cytotoxic lymphocytes, as they express high levels of intracellular perforin. In adults treated for HD, an increased activity of extrathymic T-cell differentiation may partially compensate for the loss of thymic-derived T cells.     

Increased serum IgD concentrations in patients with Hodgkin's disease

Serum IgD concentrations have been determined in twenty-one patients with Hodgkin's disease, in eight patients with non-Hodgkin's lymphomas and in twenty-eight normal (control) individuals by both a solidphase radio-immuno-assay and radial-immunodiffusion. Fourteen of the Hodgkin's patients displayed three to forty-five-fold increased serum IgD levels as compared to control individuals, while in the remaining seven patients IgD concentrations were practically normal. Patients with non-Hodgkin's lymphomas had decreased IgD concentrations.     

Lack of correlation between plaque burden and cognition in the aged monkey

The incidence rates of IDDM in Italy show remarkable variability. Sardinia, a region with the second highest incidence rate in the world, co-exists with other regions with lower rates. We review and compare epidemiologic data on the incidence of childhood-onset IDDM in Italy. papers published from 1980 to 1996 reporting incidence data in Italian areas were found by search of Medline and non-indexed Italian journals. The incidence data found cover only 57% of the Italian population. The analysis of our results shows how difficult it is to make a careful study of epidemiology of IDDM in Italy. The RIDI (the Registry for Insulin-dependent Diabetes mellitus in Italy) project started in 1996 according to international guidelines. The aims is to coordinate local IDDM registries, to promote the start of new registries in uncovered areas, and to standardize registration and data collection.     

Reed-Sternberg cells in sputum from a patient with Hodgkin''s disease. A case report

A patient with stage IV Hodgkin's disease developed severe dyspnea and was found to have pulmonary infiltrates one year following his initial diagnosis of nodular sclerosing Hodgkin's disease, stage IIB. Two separate cell block sputum cytology samples demonstrated Reed-Sternberg cells. The patient died of respiratory failure. At autopsy the main findings were extensive confluent nodules of Hodgkin's disease nearly replacing the pulmonary parenchyma of both lungs. Cytology of sputum or bronchial washings may reveal diagnostic Reed-Sternberg cells and establish the diagnosis of pulmonary Hodgkin's disease without a lung biopsy.     

Lack of involvement of known oncogenic DNA viruses in Epstein-Barr virus-negative Hodgkin's disease

Epstein-Barr virus (EBV) is associated with around one-third of cases, but young adult cases are rarely EBV associated. In this study, known oncogenic DNA viruses, including human adenoviruses, papovaviruses and the human herpesviruses-6 (HHV-6) and -8 (HHV-8) were not detected in Hodgkin's disease lesions. These results suggest that an as yet unidentified infectious agent is involved in the pathogenesis of non-EBV-associated Hodgkin's disease.     

Lack of correlation between apoptosis and DNA single-strand breaks in X-irradiated human peripheral blood mononuclear cells in the course of ageing

Inositol(1,3,4,5)tetrakisphosphate (InsP4) and phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42IP4 previously purified from pig cerebellum, which binds InsP4 (Kd approximately 2 nM) and PtdInsP3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42IP4 (pig) is highly homologous to centaurin-alpha, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42IP4 is expressed in rat brain and how it might be related to centaurin-alpha. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42IP4, but not to centaurin-alpha. The existence of p42IP4 in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-alpha-like protein in rat brain; and (3) the amino acid sequences deduced from p42IP4 cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42IP4 from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.