Latent and replicating forms of Epstein-Barr virus DNA in lymphomas and lymphoproliferative diseases

Epstein-Barr virus (EBV) is associated with lymphomas and lymphoproliferative diseases that occur mainly in immunocompromised patients, but the role EBV plays in their pathogenesis is unclear. The evidence linking EBV etiologically to these disorders includes the presence of EBV DNA and nuclear antigens in the lesions and serologic evidence that some patients with these lesions are experiencing primary or reactivated EBV infections. These syndromes may represent proliferation of cells latently infected with EBV, but the possibility of viral replication has not been rigorously studied. DNA extracted from biopsies of 35 lymphoproliferative diseases was probed with regions of the EBV genome capable of distinguishing circular, episomal DNA found in latency from linear, replicating EBV DNA. All samples contained restriction fragments characteristic of fused termini, indicative of circular, latent genomes. Thirteen samples contained additional restriction fragments diagnostic of linear EBV DNA. Therefore, replicating EBV DNA is found in approximately 40% of EBV-associated lymphoproliferative disorders.     

双探针原位杂交法检测慢性肝病和肝细胞癌患者肝组织TTV DNA的感染状况

目的 探讨慢性肝病和肝细胞癌患者肝组织TIV DNA的感染状况。方法采用PCR扩增法分别合成G1a、G2b两种亚型的双链TTV DNA探针。应用两型探针对45例肝组织标本进行TTV DNA原位杂交检测，巢氏PCR法检测血清TTV DNA。结果31例血清TTTV DNA阳性患者的肝组织TTV DNA均为阳性(100％)。14例血清TTV DNA阴性的患者肝组织中TTV DNA阳性者7例(50%)。慢性肝病患者的肝组织中TTV DNA散在分布在汇管区周围的肝细胞核内，肝癌患者TTV DNA则集中分布在肝癌细胞核内及癌组织周围的肝细胞核内。结论慢性肝病与肝癌患者肝组织中TTV DNA的感染状态存在一定差异。     

Failure to detect Epstein-Barr virus DNA in peripheral blood mononuclear cells of most patients with large granular lymphocyte leukemia

Clonal disease of large granular lymphocytes (LGLs) may arise from either CD3+ LGLs (LGL leukemia) or CD3- LGLs (natural killer [NK] cell leukemia). Other patients have chronic LGL proliferations that cannot be proven to be clonal (lymphoproliferative disease of granular lymphocytes [LDGL]). It was recently shown that clonally expanded CD3- LGLs from Japanese patients contain Epstein-Barr virus (EBV) DNA sequences, arguing for a direct causative role for EBV in NK cell leukemia. The aggressive clinical course and other clinical features of these Japanese patients differ markedly from the clinical features of LGL leukemia and CD3- LDGL patients in the United States and Europe, suggesting different pathogenic mechanisms. Therefore, we performed serologic and DNA hybridization studies for EBV in 31 patients from the United States and Europe (18 with LGL leukemia and 13 with chronic CD3- LDGL). All patients had serologic evidence for past infection with EBV. We did not detect EBV DNA sequences in peripheral blood mononuclear cell DNA from any of these patients in Southern blot hybridization analyses. EBV DNA sequences were detected after polymerase chain reaction amplification of peripheral blood mononuclear cell DNA in only 2 of 18 LGL leukemia patients and 4 of 13 chronic CD3- LDGL patients. These results argue against a direct causative role for EBV infection in LGL leukemia or chronic CD3- LDGL occurring in the United States and Europe.     

Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA.

We previously found that a form of Epstein-Barr virus with rearranged DNA induces replication of latent Epstein-Barr virus. We now have found that one of three fragments of this rearranged DNA, when cloned in recombinant plasmids and used to transfect cells, can activate expression of several polypeptides from a latent viral genome. The 33-kDa protein that is the product of the active fragment is likely to be responsible for disruption of latency.     

Mechanisms of thrombocytopenia in patients with lymphoproliferative diseases.

Different factors are involved in the development of thrombocytopenia in patients with lymphoproliferative disorders. Significant correlation was detected between the number of megakaryocytes in bone marrow and platelet count (r = 0.485, p = 0.002, n = 37) and significant difference between the number of megakaryocyte in patients with normal platelet count (> 200,000/microliters) and patients with marked thrombocytopenia (platelet count < 100,000/microliters). All patients in the latter group (n = 15) had a relatively low number of megakaryocytes. Low but significant reverse correlation was found between the level of platelet-associated IgG (PA-IgG) and platelet count (r = -0.249, p = 0.024, n = 82) and significant difference between the mean levels of PA-IgG in the groups of patients with platelet count > 200,000/microliters and < 100,000/microliters. PA-IgG were increased in 46% of patients in the total group and in 65% of patients with platelet count < 100,000/microliters. The correlation between platelet count and PA-IgG was about 2 times higher in splenectomized (r = -0.549, p = 0.005, n = 24) than nonsplenectomized patients. All splenectomized patients with platelet count < 100,000/microliters (n = 8) had a significant increase in PA-IgG. Serum antibodies were detected in only 7% of tested patients. This group was characterized by severe thrombocytopenia (in 6 of 10 patients--platelet count < 50,000/microliters) and a high incidence of haemorrhages (in 5 of 10 patients). Thus the depression of platelet production was suggested to be the basic cause of thrombocytopenia in lymphoproliferative disorders. Involvement of immune mechanisms was revealed in a large number of patients and correlated with a deeper and more complicated thrombocytopenia.     

Use of monosomics to map cloned DNA fragments in maize

Monosomic maize (Zea mays L.) plants were generated using the r-X1 deficiency system, and the monosomy was confirmed both genetically and cytologically. Genomic DNAs prepared from a group of plants, each monosomic for one chromosome, were digested with restriction enzymes, electrophoresed in agarose gels, and blotted onto nylon membranes. Hybridization of labeled cloned DNA fragments to these blots proved efficient in assigning each fragment to the chromosome from which it originated. Cloned DNA has previously contributed to loci detection through the use of the restriction fragment length polymorphisms (RFLPs), these loci subsequently being arranged into linkage groups by segregation analysis. In this study, these linkage groups were assigned to specific chromosomes, facilitating the construction of a linkage map for maize containing 112 RFLP loci. An additional 35 loci were also assigned to chromosomes by this method; however, the linkage relationships of these loci to other RFLP loci on each chromosome remains undetermined.     

Invasive pulmonary aspergillosis in patients with neoplastic diseases

Invasive pulmonary aspergillosis is an important cause of morbidity and mortality in granulocytopenic patients. The purpose of this article is to review the current understanding of the microbiology, hospital epidemiology, clinical manifestations, diagnosis, prevention, and treatment of invasive pulmonary aspergillosis. Aspergillus conidia (spores) are inhaled from environmental sources into the paranasal sinuses and lower respiratory tract. Persistent fever, pulmonary infiltrates, and pleuritic pain in granulocytopenic patients receiving antibacterial antibiotics is a common manifestation of invasive pulmonary aspergillosis. Computerized tomographic scans of the chest often reveal characteristic peripheral nodules that also may progress to characteristic cavitary lesions. Hemoptysis may develop due either to hemorrhagic infarction during granulocytopenia or to the rupture of mycotic aneurysms during recovery from granulocytopenia. Aspergillus organisms may extend locally from the lung to involve other thoracic structures, including the heart and chest wall, and may disseminate to extrapulmonary sites, such as the brain, where focal neurological deficits ensue. Early diagnosis of invasive pulmonary aspergillosis may be difficult. Isolation of Aspergillus organisms from respiratory secretions of a persistently febrile granulocytopenic patient is usually indicative of invasive pulmonary aspergillosis and should not be dismissed as a contaminant or saprophyte. Amphotericin B is the treatment of choice; however, high dosages (1.0 to 1.5 mg/kg/day) are often necessary. Aspergillosis may develop in granulocytopenic patients who are already receiving empirical amphotericin B in lower doses (0.5 to 0.6 mg/kg/day). It is hoped that further investigation directed toward an understanding of pathogenesis, improving diagnostic methodology, and developing new therapeutic and preventive strategies will improve the outcome of this life-threatening infection.     

Cardiac transplantation in patients with preexisting neoplastic diseases

Cardiac transplantation has traditionally been reserved for individuals with end-stage congestive heart failure (CHF) in whom there is no history of other life-threatening systemic disorders. In most transplant centers, patients with a history of malignancy and severe heart failure have not been considered acceptable candidates for cardiac transplantation. In the last 4 years at Stanford University Medical Center, 8 cardiac transplants have been performed in 7 patients with a history of neoplastic disease. Six of these patients had already received treatment for lymphoproliferative disorders and in 1 case, a patient underwent a transplant after treatment for adenocarcinoma of the colon. Six of the 7 patients were discharged from the hospital and in that group, the 1-year posttransplant survival rate was 71%. This was comparable to an overall 1-year survival rate of 80% for patients undergoing a cardiac transplant at our center during the same period of time. At follow-up averaging over 2 years, there has been 1 case of recurrent neoplasia. One patient with evidence of radiation-induced pulmonary damage died of respiratory failure 2 days after transplantation. One patient required retransplantation because of intractable rejection and subsequently died from infectious complications. Immunosuppressive therapy in these patients has not been associated with an increased risk for neoplastic recurrence or for the development of posttransplant lymphoproliferative disorders. The current study demonstrates that in a carefully selected group, previously treated neoplastic disease should not represent a contraindication to cardiac transplantation.     

Autoimmune liver disease in patients with neoplastic diseases

BACKGROUND: Development of de novo autoimmune liver disease has not been well documented in patients with malignant diseases. METHODS/RESULTS: In this paper we report on a series of six patients with neoplastic disorders who acquired liver disease with autoimmune features. Five patients had suffered from haematological neoplasms and one from colonic cancer. In two patients, liver disease was detected at the time of presentation with malignancy. In the remaining four, all of whom were successfully treated for malignancies, features of liver disease presented at intervals 24-72 months after the cancer diagnosis. Twelve liver specimens (11 biopsies and one hepatectomy specimen) were obtained at time intervals of 1-76 months after initial presentation of neoplastic disease. Biopsies from three patients showed features of hepatitis (one acute, one sub-acute, one chronic). Two patients had histological features suggestive of an overlap syndrome (one autoimmune hepatitis/primary biliary cirrhosis, one autoimmune hepatitis/primary sclerosing cholangitis). The sixth patient had features of autoimmune cholangiopathy. All but one responded well to steroid therapy with complete clinical and biochemical remission obtained 4 weeks to 8 months after steroid introduction. We discuss briefly possible aetiologies of autoimmune liver disease in these patients. CONCLUSIONS: Autoimmune liver disease may be precipitated by therapy for neoplastic disease or malignant disease itself. The unusually heterogeneous clinicopathological findings in this group as well as the response to treatment support the concept of a wide spectrum of manifestations of autoimmune liver disease. The results may also suggest that autoimmune liver disease may be possibly added to the list of paraneoplastic syndromes. Further prospective studies are required to confirm a causal association and to determine whether the mechanisms involved are disease- or treatment-related.     

Detection of Epstein-Barr virus DNA in hepatocellular carcinoma tissues from hepatitis C-positive patients

BACKGROUND: We have previously shown that hepatitis C virus (HCV) replication is promoted by the Epstein-Barr virus (EBV) in vitro. The aim of this study was to examine the EBV load in hepatocellular carcinoma (HCC) tissues from HCV antibody-positive patients. METHODS: DNA was extracted from paraffin sections from 168 HCC patients. After amplification of a region in the EBV BamHI W sequence by means of the polymerase chain reaction (PCR), it was detected by Southern hybridization and semi-quantified. Ten hyperplastic lesions from HCV-positive patients and 35 non-tumorous samples from hepatitis-negative patients served as controls. The PCR results were analyzed on the basis of the patient's hepatitis status. Univariate and multivariate analyses were performed to identify clinicopathologic factors for predicting EBV infection in HCC tissues. RESULTS: More than one copy of EBV DNA per 100 cells was detected in 56 (33%) of the HCC sections. The detection ratio in HCC tissues from HCV antibody-positive patients was 40% (45 of 113), which was significantly higher than that in tissues from HBV surface antigen-positive patients (14%, 5 of 37; P = 0.0018). The patient's serum HBV surface antigen and HCV antibody independently predicted the EBV positivity of HCC tissues. CONCLUSIONS: These results support our hypothesis that EBV could play an important role in the development of HCV-related HCC.     

The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes

A plasmid DNA library was constructed from restriction endonuclease digested genomic deoxyribonucleic acid (DNA) of a virulent strain of Mycobacterium tuberculosis isolated from sputum of a patient. The sensitivity and specificity of two of the cloned DNA fragments in detecting M. tuberculosis and its related DNA sequences were analysed by DNA-to-DNA hybridization. The level of detection was determined to be 50 picograms of M. tuberculosis DNA, which is approximately equivalent to 10,000 mycobacterial genomes. These two M. tuberculosis DNA probes did not cross-hybridize to DNA of non-mycobacterial origin, nor with DNA from 9 out of 11 other mycobacterial species. Mycobacterial DNA sequences could be detected in 134 of 441, or 30.4%, of various types of uncultured clinical specimens from 365 patients by the DNA probes, whereas traditional culture method showed only a 19.0% positivity rate for the same specimens (p less than 0.001). The overall sensitivity and specificity of the DNA probes in detecting M. tuberculosis are 90.5% and 83.8% respectively. The DNA hybridization test may become a useful tool for the early and rapid determination of mycobacterial infection in uncultured clinical specimens.     

Nonbacterial thrombotic endocarditis in patients with malignant neoplastic diseases

Seventy-five patients with malignant neoplastic disease and nonbacterial thrombotic endocarditis (NBTE) were studied. The over-all frequency of NBTE was double that observed in other reported autopsy series not limited to patients with cancer. The incidence in autopsy patients with bronchiolar and adenocarcinoma of the lung was twice that of patients with pancreatic and prostatic adenocarcinoma and seven times that of patients with breast cancer. The development of NBTE could not be ascribed to duration of illness or nutritional state. Since 14 patients died of cerebral infarcts and five had major myocardial infarcts resulting from thromboemboli, these and other complications of NBTE should be anticipated particularly in patients with those cancers most often associated with vegetative endocarditis.     

Failure to detect Epstein-Barr virus (EBV) DNA in plasma by real-time PCR in a case of EBV-associated posttransplantation lymphoproliferative disorder confined to the central nervous system

We report here a patient who developed multiple central nervous system (CNS) space-occupying lesions 6 months after bone marrow transplantation from an HLA-matched unrelated donor. He had extensive chronic graft-versus-host disease and severe thrombocytopenia. Posttransplantation lymphoproliferative disorder (PTLD) was diagnosed after biopsy of the lesion was facilitated by the transfusion of 40 units of platelets. Epstein-Barr virus (EBV) DNA was not initially detected in the peripheral blood by real-time polymerase chain reaction, and the blood became positive for EBV at a low level only after more than 6 weeks had passed since the initial identification of detectable intracranial lesions. The patient died of cerebral herniation while donor leukocyte infusion was being prepared, and an autopsy confirmed the diagnosis of EBV-associated PTLD restricted to the CNS.     

Use of DNA probes to detect Mycobacterium intracellulare and "X" mycobacteria among clinical isolates of Mycobacterium avium complex.

A mycobacterial DNA probe (designated X) was recently developed to help identify Mycobacterium avium complex (MAC) isolates that are nonreactive with probes specific for M. avium or Mycobacterium intracellulare. The prevalence of X probe-positive mycobacteria in clinical specimens and their role in causing disease is unknown. Using a DNA probe kit that includes the X probe, we characterized 100 consecutive clinical MAC isolates as M. avium, M. intracellulare, or X. Lysates from 81 of the isolates reacted with the M. avium probe, 13 with the M. intracellulare probe, 3 with the X probe, and 3 failed to hybridize with any of the probes. All three X-positive isolates were recovered from sputa of patients who were recent immigrants to the United States and who presented with hemoptysis. One isolate was from a Hispanic man infected with human immunodeficiency virus type 1 (HIV-1) and the other 2 were from Filipino patients with no HIV-1 risk factors. This study also showed a higher than expected number of M. intracellulare isolates from blood and cerebrospinal fluid of HIV-1-infected patients.     

Adverse effects of thalidomide administration in patients with neoplastic diseases

Thalidomide, a glutamic acid derivative, was withdrawn from clinical use in 1962 due to its severe teratogenic effects. Its recent reinstitution in clinical practice was related to its benefits in leprosy and multiple myeloma. Moreover, the antiangiogenic and immunomodulatory properties of thalidomide have led to its evaluation in several malignant diseases, including myelofibrosis, renal cell cancer, prostate cancer, and Kaposi sarcoma. However, thalidomide use is associated with several side effects: somnolence and constipation are the most common, while deep vein thrombosis and peripheral neuropathy are the most serious. A combination of thalidomide with steroids or chemotherapy is being evaluated in several phase 2 studies. While it is not yet clear whether these combinations will enhance efficacy, they appear to increase the toxicity of thalidomide, and thalidomide analogs are being developed to minimize this toxicity. Ongoing studies will clarify the potential advantages of these agents in the treatment of neoplastic diseases.