Epstein-barr virus-negative human malignant T-cell lines.

Two lymphoblastoid lines, CCRF-CEM and HSB-2, with properties of malignant cells, derived from children with leukemia secondary to lymphosarcoma, have T-cell properties and lack Epstein-Barr virus antigens.

HTLV-I--associated B-cell CLL: indirect role for retrovirus in leukemogenesis.

Serum containing antibodies to the human T-lymphotropic virus type I (HTLV-I) has been observed at a higher than expected frequency in patients with B-cell chronic lymphocytic leukemia (CLL) in an area endemic for HTLV-I. An attempt was made to determine whether the cells from patients with this leukemia were HTLV-I antigen-committed B cells that had undergone malignant transformation. Cells from two HTLV-I seropositive Jamaican patients with CLL were fused with a human B-lymphoblastoid cell line. The hybridoma cells that resulted from the fusion of CLL cells from patient I.C. produced an immunoglobulin (IgM) that reacted with the p24 gag protein from HTLV-I, HTLV-II, and HTLV-III (now referred to as HIV), but showed preferential reactivity with HTLV-I. The specific immunoglobulin gene rearrangement (IgM, kappa) in the CLL cell was demonstrated in the hybridoma cell line, indicating that the captured immunoglobulin was from the CLL cells. The IgM secreted by the fusion of CLL cells from patient L.L. reacted only with HTLV-I-infected cells and with the HTLV-I large envelope protein (gp61) on Western blots. The CLL cells from these patients appear to be a malignant transformation of an antigen-committed B cell responding to HTLV-I infection, suggesting an indirect role for this retrovirus in leukemogenesis.

Alkaline phosphatase in Epstein-Barr viral nuclear antigen--positive cell lines.

The production and nature of alkaline phosphatase were studied in Epstein-Barr viral nuclear antigen-positive, surface membrane immunoglobulin negative-cell lines established from two patients, one with acute myeloid leukemia and one with acute lymphoblastic leukemia. The acute myeloid leukemia-derived cells contained myeloid alkaline phosphatase, while the acute lymphoblastic leukemia-derived cells contained lymphoid alkaline phosphatase. The presence of the myeloid-specific enzyme in a surface membrane immunoglobin--negative cell line suggests that the line is composed of myeloid precursor cells and that such cells may be susceptible to infection with Epstein-Barr virus.

Myeloma, Hodgkin disease, and lymphoid leukemia after renal transplantation: characteristics, risk factors and prognosis.

BACKGROUND: Hodgkin disease and myeloma were recently included in the classification of posttransplant lymphoproliferative disorder (PTLD). However, because their incidence is low, not much is known about their particular features. METHODS: The incidence, characteristics, risk, and prognostic factors of myeloma, Hodgkin disease, and lymphoid leukemia using the United States Renal Data System from 1991 to 2000 among 66,159 Medicare patients were analyzed. RESULTS: In all, 1,169 recipients developed a lymphoid disease: 823 (1.2%) non-Hodgkin's lymphomas (NHL), 160 (0.24%) myelomas, 60 (0.1%) Hodgkin lymphomas, and 126 (0.2%) lymphoid leukemias. Older age was associated with an increased risk of myeloma and leukemia. The incidence of hepatitis C virus infection was higher in recipients with myeloma (6.9 vs. 3.9%, P=0.05). Induction therapy was associated with a greater risk of myeloma and leukemia, but not Hodgkin disease. Azathioprine was associated with a lower risk of myeloma, and tacrolimus with a lower risk of Hodgkin disease. According to the type of malignancy, ten-year survival rates were significantly different: 42, 26, 55 and 39% respectively for NHL, myeloma, Hodgkin disease, and leukemia. CONCLUSION: These results support specific features and risk factors related to the occurrence of each type of lymphoid-proliferation and suggest for the first time a possible association between hepatitis C virus and myeloma in kidney transplant recipients.

Lymphoproliferative diseases of fowl: JM-V leukemic lymphoblasts in cell culture.

JM-V leukemic lymphoblasts were established in cell culture. The cultured cells (JM-VLC cells) were transplantable in young chicks and produced a disease indistinguishable from JM-V lymphoblastic leukemia as initiated by whole-blood inoculation. JM-VLC cells maintained a normal female karyotype through 13 passages in Rhode Island Red cockerels. With the use of JM-V antisera and antisera from birds with naturally occurring Marek's disease (MD), specific antigens were detected on the surfaces of living cells. Intracellular antigens were detected with anti-MD virus sera after cultivation for at least 1 day at 37 degrees C. In spite of the expression of MD antigens, the presence of herpesvirus particles associated with the cultured cells, and the occurrence of foci of multinucleated cells in kidney cultures from chicks inoculated with cellfree preparations of JM-VLC cells, the pathologic potential of the cultured cells was that of JM-V leukemia.

Establishment of an Epstein-Barr virus-determined nuclear antigen-negative human B-cell line from acute lymphoblastic leukemia.

A human B-cell line designated as BALL-1 was established from the peripheral blood of a patient with acute lymphoblastic leukemia (ALL). Neither Epstein-Barr virus (EBV) particles nor EBV-determined nuclear antigen (EBNA) was detectable. The morphologic and growth characteristics were clearly distinct from those of numerous EBV-positive lymphoblastoid cell lines previously reported. BALL-1 cells probably originated from the donor's leukemia cells as judged from their cytogenetic, morphologic, and surface features. The BALL-1 line was the first EBNA-negative B-cell line established from ALL.

Biologic properties of viable deletion mutants of simian virus 40 (SV40) rescued from the cells of an SV40-induced hamster lymphocytic leukemia.

A lymphocytic leukemia induced by the oncogenic DNA simian virus 40 (SV40) in an inbred LSH/SsLak Syrian golden hamster was evoked to produce infectious SV40 by fusion of the leukemia cells with grivet monkey kidney (GMK) cells and by exposure of the leukemia cells to the chemical inducers mitomycin C and cycloheximide. Plaque-purified viable substrains of the rescued SV40 when studied by restriction endonuclease digestion of viral DNA were found to contain small deletions within the Hind III restriction fragment C. These deletions lay near the viral origin of DNA replication. Ten plaque-purified substrains of the rescued virus identified by immunofluorescence as being SV40 were found, when compared to the wild-type SV40, to replicate slowly and to form small plaques. Although these substrains transformed NIH/3T3 cells as efficiently as the wild-type SV40 in tissue culture, they were generally less oncogenic in vivo--7 of the 10 failed to induce tumors. The 3 oncogenic SV40-rescued substrains were not found to exhibit "lymphocytotropism," i.e., the capacity to infect and neoplastically transform preferentially hamster lymphocytes. Thus the hamster lymphocytic leukemia originally induced by the wild-type SV40 was most likely a chance-stochastic event rather than the result of tropism-determinism mediated by the virus, as is usually the case with leukemogenic RNA viruses.

Characterization of Epstein-Barr virus-carrying cell lines established from chronic lymphocytic leukemia.

Attempts were made to establish lymphoid cell lines from the cultured peripheral blood lymphocytes of six patients with chronic lymphocytic leukemia. In only one case was cell growth obtained following the addition of exogenous transforming Epstein-Barr virus, and those cell cultures proved not to have acquired the ability to proliferate permanently. In the same case, cell lines were established spontaneously from the peripheral blood without addition of Epstein-Barr virus. The cells which grew spontaneously were large, were occasionally weakly surface adherent, and grew in suspension as loose clumps or as single cells. They were negative for surface immunoglobulins and spontaneous rosette formation with sheep erythrocytes and positive for intracytoplasmic immunoglobulins (Fc and C3 receptors). At an early passage, the spontaneous lines had an aneuploid karyotype with some triploid and some tetraploid cells. Structural chromosomal aberrations include a 14q+. Electron microscopy of the chronic lymphocytic leukemia lines revealed relatively smooth surfaces with numerous mitochondria, widespread vacuolization, and numerous unusual "myelin" figures. Five to 10% of the cells were phagocytic as detected by internalization of latex particles; however, they were Epstein-Barr nuclear antigen positive. The nature of these cells and their possible relationship to the etiology of chronic lymphocytic leukemia are discussed.

Induction of permanently proliferating human lymphoblastoid lines by N-methyl-N'-nitro-N-nitrosoguanidine.

N-Methyl-N'-nitro-N-nitrosoguanidine treatment of human peripheral blood mononuclear cells increases the frequency of lymphocyte transformation into permanently proliferating lines. The transformation is dependent on the dose of N-methyl-N-7-nitro-N-nitrosoguanidine and does not occur in the presence of autologous human serum. Thirty-eight of the forty-two established lines resulting fron N-methyl-N'-nitro-N-nitrosoguanidine treatment produced detectable Epstein-Barr virus antigens.

Cell-surface antigens induced by Friend and Rauscher virus complexes and their associated lymphatic leukemia viruses in the rat.

The WKA/Mk rat tumors induced by Friend virus complex, Rauscher virus complex, and their associated lymphatic leukemia viruses were investigated for their antigenic relationhips with transplantation experiments and cytotoxicity tests. It was found that Friend lymphatic leukemia virus-induced tumors lacked part of the tumor-associated transplantation antigens (TATA's) on Friend virus complex-induced tumors, and the former did not express the type-specific (Friend) TATA for the latter not shared by Rauscher virus complex-induced tumors, which was previously reported by the authors. In contrast, the antigenic differences between TATA's of Rauscher virus complex-induced tumors and those of Rauscher lymphatic leukemia virus-induced tumors were not clearly demonstrated. Furthermore, these studies indicated that Rauscher lymphatic leukemia virus-induced tumors had a weak type-specific TATA not shared by the tumors induced by Friend lymphatic leukemia virus. These results of transplantation studies were also serologically supported by cytotoxicity tests.

Biochemical approaches to detection of Epstein-Barr virus in human tumors.

The application of biochemical studies for the detection of Epstein-Barr virus (EBV)-DNA in human tumor cells is discussed. These studies resulted in the consistent demonstration of viral nucleic acid in African Burkitt's lymphoma biopsies and in epithelial tumor cells of nasopharyngeal carcinomas. The viral DNA resides within those cells regularly in multiple copies per cell. Besides these tumors our group detected significant concentrations of EBV-DNA in a German lymphoma patient revealing histological characteristics of Burkitt's lymphoma. Moreover, virus DNA was also found in a patient suffering from immunoblastic lymphadenopathy. More than 50 additional B-cell lymphomas and more than 40 biopsies from patients with Hodgkin's disease did not contain detectable amounts of EBV-DNA when tested by nucleic acid hybridization. A tentative scheme of EBV-induced pathogenesis is discussed.

IgG-subclass-specific CMV reactivity in bone marrow transplant recipients.

IgG subclasses of cytomegalovirus (CMV) antiviral antibodies were determined in 37 donor-recipient pairs of bone marrow transplants (BMT). Bone marrow transplant recipients, like healthy persons, have a restricted immune reactivity, producing mainly two types of anti-CMV IgG: IgG1 and IgG3. Passively transfused specific antibody subclasses were readily measurable. Take of the transplant could be detected from the production of subclass IgG antiviral antibody 1-3 months after BMT of seronegative recipients with marrow from seropositive donors. Patients with protracted CMV infections or other severe diseases initially also produced CMV IgG1 and IgG3, but anti-CMV subclass titers then decreased. In severe disease, CMV was isolated from blood cells as well as from urine. In moderate infections, in which the patients recovered, CMV was isolated from urine but usually not from blood, and a strong and durable antiviral subclass response was measured.

Comparison of the tissue distribution of reverse transcriptase, p30 and type-C virus in a gibbon ape with lymphocytic leukemia.

Tissues obtained at necropsy from a 7 year old male gibbon ape with malignant lymphoma and leukemia were analyzed by electron microscopic, immunological and enzymological techniques to determine the comparative localization of tumor cells and virus throughout the body. In general, the different assays correlated well; the reverse transcriptase (RT) assay and p30 radioimmunoassay (RIA) being the most sensitive, although the RT assay was able to detect activity in one tissue scored negative by p30 RIA. Tissues were infiltrated with tumor cells to varying degrees which correlated well with the level of virus markers in most cases with the exception of the liver and kidney. In these 2 organs there was marked infiltration of free virus and tumor cells but there was no evidence of virus infection or production by these cells.

Phenotypic and functional characterization of HTLV positive neoplastic T cells cultured with interleukin-2--I. Retention of morphology, phenotype and selective cytotoxic properties in long term culture.

T cells from the peripheral blood of a T-cell chronic lymphocytic leukemia (T-CLL) patient, cultured in the presence of interleukin-2 (IL-2), were found to express the p19 structural core protein of the human T-cell leukemia/lymphoma virus (HTLV) and to release type C virus particles. Comparison of the T-CLL cell line with the original leukemic T cells revealed that both the fresh and the proliferating T-CLL cells were pleomorphic cells that showed a convoluted nucleus and formed rosettes with sheep erythrocytes (E-rosettes). They were reactive with the monoclonal antibodies OKT1, OKT4 and OKT11, but not with OKT3, OKT6 or OKT8, indicating that they were mature T cells but that they differed from normal T cells in their lack of reactivity with OKT3. In addition they did not bind peanut agglutinin or OKM-1, and were negative for Epstein-Barr nuclear antigen, surface immunoglobulin, non-specific esterase activity of Fc- or complement receptors. Part of the fresh T-CLL cells reacted with a monoclonal antibody recognizing HLA-DR antigens (p29, 34) (36%) and with anti-Tac (62%), a monoclonal antibody directed at the IL-2 receptor, indicating that the T-CLL cells were partially activated already in vivo. After culture in vitro all proliferating T-CLL cells expressed HLA-DR and Tac antigens. The fresh T-CLL cells were found to be defective in cell-mediated lympholysis (CML) generated in mixed lymphocyte culture (MLC), antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). In addition they failed to exhibit natural killer (NK) cell activity against targets that are usually very susceptible to lysis, such as K562, but were able to kill two tumor-derived cell lines, the melanoma NKI-4 and the neuroblastoma CHP-100. The same pattern of selective killing was observed using the proliferating T-CLL cells as effectors, or cloned T-CLL cultures obtained from them by limiting dilution procedures. Therefore, it was concluded that the T-CLL cells represented a clonal expansion of neoplastic T cells that retained their phenotype and cytotoxic properties after culture in vitro.

Demonstration of terminal deoxynucleotidyl transferase in single cells by indirect immunofluorescence. II. An examination of specificity.

Advantages in the use of indirect immunofluorescence for the identification of terminal transferase (TdT) in single cells may be offset by lack of specificity, as compared to the biochemical assay of the enzyme, especially in analyses of lymphocyte populations. False positive results were obtained in 15/15 tonsillectomy samples and in 9/27 specimens from children with acute lymphoblastic leukemia in remission, perhaps due to antihuman lymphocyte activity in the rabbit heteroantisera which are used in the indirect immunofluorescence technique. This phenomenon may be more pronounced in 'activated' normal lymphocytes. Such reactions are not due to antibodies directed against adenovirus, papovavirus or EB virus antigens, although these are common constitutents of human tonsillar cells. Additional problems with TdT heteroantisera may result from immunization with non-TdT determinants in calf thymus extracts, as was manifest in human non-lymphoid (KB) cells cultured in fetal bovine serum. These difficulties will be overcome only by production of a monoclonal antibody using human TdT as the antigen.

In-vitro infection of chronic lymphocytic leukemia cells by Epstein-Barr virus (EBV).

We sought to determine the potential of infecting lymphoid cells from patients with chronic leukemia (CLL) with Epstein-Barr virus (EBV) by testing for EBV receptors (EBVR) by flow cytometry, assessing for infectability of these cells by culturing with B95-8-derived virus, and staining for EB nuclear-associated antigens (EBNA) at various times post-infection. EBVR were present on 54-91% of lymphoid cells in seven cases of CLL and on 46% of prolymphocytic leukemia cells. Dynamic changes regarding EBNA positivity, morphology, and viability occurred post-infection with the virus. On day 2 only a few EBNA-positive lymphoblasts were observed. On days 11-21 positivity increased from 2 to 34% of cells. Simultaneously, the viable cell number declined to approximately 1/10th of original number. A significant proportion of the EBNA-positive cells corresponded to the original CLL cells. In 3 of 7 cases of CLL a Pan T-cell phenotype was demonstrated by Leu-1 monoclonal antibody testing. The infected cells did not react with two monoclonal antibodies, EBV-CS 1 and 4, which react with B-cell lymphoblastoid cell lines (B-LCL). Moreover, the B-LCL derived at 1-2 months post-infection of CLL cells did not express the Leu-1 antigen, but expressed EBV-CS 1 or 4 defined antigens. In the prolymphocytic leukemia, 64% of the cells showed EBNA positivity on day 7 and giant cells with huge round or multiple nuclei appeared which were EBNA-positive. CLL and prolymphocytic leukemia cells can be infected as demonstrated by EBNA-positivity. This infection does not lead to immediate transformation, but evokes lymphoblast and multinucleated giant cell production prior to the death of cells.

Epstein-Barr virus-associated natural killer-large granular lymphocyte leukemia.

We describe the first case of an Epstein-Barr virus (EBV)-associated natural killer-large granular lymphocyte (NK-LGL) leukemia in the United States to the best of our knowledge. A 29-year-old woman of Japanese descent developed EBV infection after a blood transfusion as indicated by a rise in serum antibody titers. Peripheral blood and bone marrow aspirate smears demonstrated increased LGLs. Flow cytometry showed that these cells expressed NK-associated surface antigens. Cytogenetic analysis of the bone marrow aspirate showed two distinct but related clones with multiple copies of a modified 7 marker chromosome. Death followed colonic perforation. Findings at necropsy included bone marrow lymphocytosis and erythrophagocytosis, a mononucleosis-like lymphadenitis, atypical hepatitis with a mixed, predominantly T-cell infiltrate, interstitial pneumonitis, and multiorgan system vasculitis with perforation of the transverse colon. Epstein-Barr virus transcripts were identified in lymphocytes infiltrating liver and peripheral nerve by in situ hybridization. In addition, Southern blot analyses showed monoclonal bands superimposed on oligoclonal ladders of EBV termini in liver and lymph node. The identical episomal form of EBV was found in the bone marrow, lymph node, and liver. No immunoglobulin (Ig), T-cell receptor beta, or T-cell receptor gamma chain gene rearrangements were identified. These studies support the hypothesis that the LGL population was a neoplastic EBV-related clonal proliferation of NK cells.

Clostridium difficile in haematological malignancy.

Twenty patients with haematological malignancies who developed Clostridium difficile bowel infection or colonisation are described. All isolates of C difficile were toxigenic in vitro and faecal cytotoxin (toxin B) was detected in 20/26 episodes. Ten of 20 episodes with detectable faecal cytotoxin were associated with typical antibiotic associated diarrhoea. In the other 10 episodes (nine patients), there was a severe unusual illness which was associated with detection of C difficile. The unusual features of the illness were pronounced jaundice (total bilirubin greater than or equal to 44 mumol/l), abdominal pain and distension, and initial constipation followed either by diarrhoea or by large bowel stasis. Four of these patients died within seven days. Bacteraemia was often a presenting feature in neutropenic patients subsequently shown to have C difficile. This was not the case in non-neutropenic patients. Bacteraemia was commonly polymicrobial and in two cases C difficile was isolated from blood culture. The clinical implications of recognition of this atypical C difficile associated syndrome are discussed.

Evidence for HTLV-I associated with mycosis fungoides and B-cell chronic lymphocytic leukemia.

Human T-cell lymphotropic virus type I (HTLV-I) is a human retrovirus that can transform T-helper lymphocytes and is etiologically associated with adult T-cell lymphoma/leukemia. Mycosis fungoides represents a primary cutaneous lymphoma of helper T-cell origin, while chronic lymphocytic leukemia is generally considered to be a neoplastic B-lymphocyte disorder. Our patient had HTLV-I with coexistent mycosis fungoides and B-cell chronic lymphocytic leukemia. The concurrent lymphoid proliferations may represent HTLV-I-associated abnormalities of immunoregulation.

Microbiological findings in febrile neutropenia.

BACKGROUND: This study was carried out to assess the isolation rate of bacterial and fungal causative agents in Mexican neutropenic adults with hematological neoplasia. METHODS: A prospective observational survey involving 120 consecutive episodes of febrile neutropenia during 1 year was carried out. These episodes were observed in 630 patients discharged with diagnoses of leukemia or lymphoma, or after bone-marrow transplantation. RESULTS: At least one pathogen was isolated in 42 of 120 episodes (35%), and was present in 39 patients with acute myeloid leukemia (AML) (43%), acute lymphoblastic leukemia (ALL) (23%), and in patients who underwent bone-marrow transplantation (20%). Primary bacteremia was the most frequent cause of fever (24 episodes, 57%), followed by intravascular device-related infections (5 episodes, 17%), and soft-tissue infections (5 episodes, 15%). Escherichia coli (33%) was the most frequently isolated agent of primary bacteremia, followed by coagulase-negative Staphylococcus (29%), and Klebsiella oxytoca (16%). Fungal infection was responsible for five events (4%): two episodes of pneumonia (Penicillium marneffei and Aspergillus fumigatus, one event each); two cases of fungemia, one due to Candida tropicalis and one to Rhodotorula gluttinis, and one cryptococcal meningitis event. CONCLUSIONS: The isolation rate, approximately 30%, was in accordance with previous reports; similar percentages of Gram-positive and Gram-negative isolates were found. A remarkably low rate of viridans group streptococci and fungal agents was observed, despite the fact that neutropenia is the main risk factor for infection due to these agents. Studies reporting local microbiological findings are necessary because they support an antibiotic choice for prophylaxis or therapy more accurately than reports from other areas.