Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA.

AIMS: To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished. METHODS: Tissue sections from the central nervous system of infected cases were examined by light microscopy, immunohistochemistry and in situ hybridization. RESULTS: All 13 cases of EV71 encephalomyelitis collected from Asia and France invariably showed stereotyped distribution of inflammation in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus and, to a lesser extent, cerebral cortex and meninges. Anterior pons, corpus striatum, thalamus, temporal lobe, hippocampus and cerebellar cortex were always uninflamed. In contrast, the eight JE cases studied showed inflammation involving most neuronal areas of the central nervous system, including the areas that were uninflamed in EV71 encephalomyelitis. Lesions in both infections were nonspecific, consisting of perivascular and parenchymal infiltration by inflammatory cells, oedematous/necrolytic areas, microglial nodules and neuronophagia. Viral inclusions were absent. CONCLUSIONS: Immunohistochemistry and in situ hybridization assays were useful to identify the causative virus, localizing viral antigens and RNA, respectively, almost exclusively to neurones. The stereotyped distribution of inflammatory lesions in EV71 encephalomyelitis appears to be very useful to help distinguish it from JE.

High prevalence of latent tuberculosis infection in patients in end-stage renal disease on hemodialysis: Comparison of QuantiFERON-TB GOLD, ELISPOT, and tuberculin skin test.

BACKGROUND: Individuals with end-stage renal disease (ESRD) are 10- to 25-fold more likely than immunocompetent people to develop active tuberculosis (TB) and are candidates for being treated for latent TB infection (LTBI). However, diagnosis using the tuberculin skin test (TST) is doubly difficult due to cutaneous anergy and cross-reactions with Bacille-Calmette-Guérin (BCG) vaccination. MATERIALS AND METHODS: This was a prospective, doublematched, cohort study in which 32 ESRD patients and 32 age-matched, healthy controls were enrolled. The TST and two new interferon-gamma blood tests, QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB (ELISPOT), were performed. The subjects were followed up 2 years for active TB disease. ELISPOT was done in ESRD patients only. RESULTS: Compared to the healthy controls, a high prevalence of LTBI was found in the ESRD patients by TST (62.5%, 95% confidence interval [CI] 43.7-78.9), QFT-G (40.0%, 95% CI 22.7-59.4), and ELISPOT (46.9%, 95% CI 29.1-65.3). Agreement was moderate (kappa [kappa] = 0.53) for QFT-G and ELISPOT but only slight between TST and QFT-G (kappa = 0.25) and fair between TST and ELISPOT (kappa = 0.32). ESRD (p = 0.03) and diabetes mellitus (p = 0.04) were significant risk factors for QFT-G positivity on the multivariable analysis. The overall rate of active TB was 1.66 cases per 100 person-years (pys), with the rate higher in patients with ESRD (3.53 per 100 pys) and those with positive (3.40 per 100 pys) and indeterminate QFT results (30.16 per 100 pys), although the difference was not statistically significant. Sensitivity, specificity, and positive and negative predictive values of QFT-G for active TB was 100%, 62.1%, 8.3% and 100%. CONCLUSION: This pilot study is the first to compare QFT-G, ELISPOT, and TST in ESRD patients on hemodialysis and demonstrates a high prevalence of LTBI in this population. In our study, the QFT-G was the more accurate method for identifying those truly infected with Mycobacterium tuberculosis, even in BCG-vaccinated individuals.

Suppression of p38 mitogen-activated protein kinase inhibits hepatitis B virus replication in human hepatoma cell: the antiviral role of nitric oxide.

The role of the p38 mitogen-activated protein kinase (MAPK) pathway in hepatitis B virus (HBV) replication was investigated in this study. After transient transfection with HBV plasmid, p38 MAPK, but not JNK or ERK1/2, was significantly phosphorylated in human hepatoma cell Huh7. Interestingly, HBV proteins and RNA synthesis were significantly inhibited by a specific inhibitor of p38 MAPK, SB203580, in a dose-dependent manner. Intracellular core-associated DNA, extracellular virion-associated DNA and covalently closed circular DNA were also significantly inhibited by SB203580. Further results showed the antiviral role of nitric oxide (NO) on the suppression of HBV replication and downregulation of p38 MAPK phosphorylation. In conclusion, these results suggested that suppression of phosphorylation of p38 MAPK by inhibitor or NO could inhibit intracellular HBV replication.

Upregulation of tumor necrosis factor-alpha gene by Epstein-Barr virus and activation of macrophages in Epstein-Barr virus-infected T cells in the pathogenesis of hemophagocytic syndrome.

A potentially fatal hemophagocytic syndrome has been noted in patients with malignant lymphomas, particularly in EBV-infected T cell lymphoma. Cytokines, such as interferon-gamma (IFN-gamma), TNF-alpha, and IL-1alpha, are elevated in patients' sera. To verify whether infection of T cells by EBV will upregulate specific cytokine genes and subsequently activate macrophages leading to hemophagocytic syndrome, we studied the transcripts of TNF-alpha, IFN-gamma, and IL-1alpha in EBV-infected and EBV-negative lymphoma tissues. By reverse transcription PCR analysis, transcripts of TNF-alpha were detected in 8 (57%) of 14 EBV-infected T cell lymphomas, higher than that detected in EBV-negative T cell lymphoma (one of six, 17%), EBV-positive B cell lymphoma (two of five, 40%) and EBV-negative B cell lymphomas (one of seven, 14%). Transcripts of IFN-gamma were consistently detected in T cell lymphoma and occasionally in B cell lymphoma, but were independent of EBV status. IL-1alpha expression was not detectable in any category. Consistent with these in vivo observations, in vitro EBV infection of T cell lymphoma lines caused upregulation of TNF-alpha gene, and increased secretion of TNF-alpha, but not IFN-gamma or IL-1alpha. Expression of TNF-alpha, IFN-gamma, and IL-1alpha was not changed by EBV infection of B cell lymphoma lines. To identify the specific cytokine(s) responsible for macrophage activation, culture supernatants from EBV-infected T cells were cocultured with a monocytic cell line U937 for 24 h. Enhanced phagocytosis and secretion of TNF-alpha, IFN-gamma, and IL-1alpha by U937 cells were observed, and could be inhibited to a large extent by anti-TNF-alpha (70%), less effectively by anti-IFN-gamma (31%), but almost completely by the combination of anti-TNF-alpha and anti-IFN-gamma (85%). Taken together, the in vivo and in vitro observations suggest that infection of T cells by EBV selectively upregulates the TNF-alpha expression which, in combination with IFN-gamma and probably other cytokines, can activate macrophages. This study not only highlights a probable pathogenesis for virus-associated hemophagocytic syndrome, but also suggests that anti-TNF-alpha will have therapeutic potential in the context of their fatal syndrome.

Human metapneumovirus infection among children in Taiwan: a comparison of clinical manifestations with other virus-associated respiratory tract infections.

This study compared the clinical, laboratory and radiological features of infections caused by human metapneumovirus (hMPV) with other respiratory viruses. Nasopharyngeal aspirates and throat swabs were obtained from children during a 9-week period. hMPV was the virus isolated most frequently, followed by adenovirus, influenza virus A, respiratory syncytial virus and influenza virus B. hMPV-infected children were younger, and were more likely to be female, to present with feeding difficulties, a rash, tachycardia and a longer duration of fever, and to cough less frequently. Increasing interstitial infiltrates and hyperinflation were the most common radiological findings. None of the children required mechanical ventilation.

Using an integrated infection control strategy during outbreak control to minimize nosocomial infection of severe acute respiratory syndrome among healthcare workers.

Healthcare workers (HCWs) are at risk of acquiring severe acute respiratory syndrome (SARS) while caring for SARS patients. Personal protective equipment and negative pressure isolation rooms (NPIRs) have not been completely successful in protecting HCWs. We introduced an innovative, integrated infection control strategy involving triaging patients using barriers, zones of risk, and extensive installation of alcohol dispensers for glove-on hand rubbing. This integrated infection control approach was implemented at a SARS designated hospital ('study hospital') where NPIRs were not available. The number of HCWs who contracted SARS in the study hospital was compared with the number of HCWs who contracted SARS in 86 Taiwan hospitals that did not use the integrated infection control strategy. Two HCWs contracted SARS in the study hospital (0.03 cases/bed) compared with 93 HCWs in the other hospitals (0.13 cases/bed) during the same three-week period. Our strategy appeared to be effective in reducing the incidence of HCWs contracting SARS. The advantages included rapid implementation without NPIRs, flexibility to transfer patients, and re-inforcement for HCWs to comply with infection control procedures, especially handwashing. The efficacy and low cost are major advantages, especially in countries with large populations at risk and fewer economic resources.

Non-lethal Candida albicans cph1/cph1 efg1/efg1 transcription factor mutant establishing restricted zone of infection in a mouse model of systemic infection.

The cph1/cph1 efg1/efg1 Candida albicans mutant cells were non-lethal in a mouse model of systemic infection. We investigated in vivo proliferation and invasion of C. albicans cells in infected mice to elucidate the interaction between the host and the pathogen. Homogenates of kidneys from the mice infected with the wild-type and the mutant C. albicans cells yielded a mean of 2.1 x 10 7 CFU/g and 2.2 x 10 6 CFU/g, respectively. The kidneys from the mice infected with the wild-type cells showed extensive renal cortical necrosis associated with neutrophilic infiltration. There were also wild-type hyphal cells present in abundance. Hence, tubular necrosis leading to renal failure in the mice may be the cause of death. Although the cph1/cph1 efg1/efg1 mutant cells were not lethal, they were capable of establishing restricted zones of infection and colonization near the renal pelvis instead of simply being cleared by the immune system in mice.

Distinctive phorbol ester-induced morphological and surface antigen changes in mycosis fungoides, the Sézary syndrome, and adult T-cell leukemia.

Clinical and pathological studies of cutaneous T-cell lymphomas (CTCL) reveal variations in tumor cell morphology and surface membrane phenotype that are of diagnostic and prognostic importance. Our study investigates blastic transformation and surface antigen change on CTCL cells in vitro under the influence of tumor-promoting phorbol ester (TPA) and phytohemagglutinin. Both agents transformed tumor cells with cerebriform nuclei into blast cells within 5 days; however, Sézary cells were somewhat resistant to transformation with phytohemagglutinin. Multinucleated cells with prominent nucleoli resembled Reed-Sternberg cells of Hodgkin's disease. These morphological changes simulated the appearance of the aggressive tumor stage of mycosis fungoides. During blastic transformation, the erythrocyte rosette receptor was induced by TPA on sheep erythrocyte-rosette-negative Sézary cells from one patient. During the first 24 hr in vitro, Sézary and MF cells stimulated by TPA lost Leu 3a (T4) antigen while maintaining original high levels of Leu 1 antigen. In contrast, leukemia cells from patients with adult T-cell leukemia (ATL) were resistant to modulation of Leu 3a antigen by TPA; 3A1 antigen on CTCL and ATL cells was unaffected by TPA. Blastic transformation of CTCL cells was observed with both TPA and phytohemagglutinin, but helper T-cell antigen Leu 3a (T4) and erythrocyte rosette receptor changes occurred only with TPA. Thus, blastic transformation and surface differentiation were not directly related. These results provide a possible model for the study of blastic transformation and surface antigen/receptor variation in CTCL. They also may provide an independent test for the distinction of CTCL and ATL in vitro. Finally, they illustrate the relative resistance of ATL to surface antigen modulation as previously shown for Tac antigen modulation by anti-Tac antibody.

Expression of P-glycoprotein and glutathione-S-transferase in recurrent lymphomas: the possible role of Epstein-Barr virus, immunophenotypes, and other predisposing factors.

PURPOSE: We previously have reported the poor prognoses of recurrent peripheral T-cell lymphoma (PTCL) and Epstein-Barr virus (EBV)-associated PTCL (J Clin Oncol 7:725-731, 1989; Blood 77:799-808, 1991). To study the role that drug resistance plays in this scenario, we conducted a retrospective study of 23 adult patients. PATIENTS AND METHODS: All patients had recurrent lymphoma tissue available for immunophenotyping and screening for the existence of EBV DNA in tumor cells by Southern blot analysis and in situ hybridization. Expression of a multidrug resistance P-glycoprotein ([P-gp]mdr-1) and a glutathione redox cycle-related glutathione-S-transferase pi (GST-pi) was determined by an immunohistochemistry method. RESULTS: Expression of mdr-1 or GST-pi was found in 11 (48%) and 12 (52%) cases, respectively. Most (11 of 12) of the GST-pi expression occurred simultaneously with mdr-1. Prechemotherapy tumor tissues were available in 11 cases; only two (18.2%) of these cases expressed mdr-1. Four (36%) of 11 cases that expressed mdr-1 (mdr-1(+)) and nine (90%) of 10 cases that did not express mdr-1 (mdr-1(-)) responded to second-line chemotherapy (P < .05). The survival-after-recurrence (SAR) curves significantly favored mdr-1(-) recurrent lymphoma (P < .05). The mdr-1 expression was further correlated with the immunophenotype and EBV association. All six cases of EBV-associated lymphoma (PTCL, five cases; Hodgkin's disease, one case) had significant simultaneous expression of mdr-1 and GST-pi in their recurrent tumor tissues. CONCLUSION: (1) mdr-1 expression is a significant prognostic factor in recurrent lymphomas; (2) high expression of mdr-1 is observed in recurrent EBV-associated PTCL; and (3) GST-pi usually expresses simultaneously with mdr-1 in recurrent lymphomas. The role of EBV in the development of mdr-1 and the biologic significance of the simultaneous expression of mdr-1 and GST-pi in recurrent lymphomas are well worth further exploration.

Prospective study of Helicobacter pylori eradication therapy in stage I(E) high-grade mucosa-associated lymphoid tissue lymphoma of the stomach.

PURPOSE: High-grade mucosa-associated lymphoid tissue (MALT) lymphomas of the stomach are generally believed to be Helicobacter pylori-independent, autonomously growing tumors. However, anecdotal cases of regression of high-grade lymphomas after the cure of H pylori infection had been described. The present prospective study was conducted to evaluate the effect of anti-H pylori therapy in stage I(E) high-grade gastric MALT lymphomas. PATIENTS AND METHODS: Sixteen patients with H pylori infection and stage I(E) gastric high-grade MALT lymphoma consented to a brief antibiotic therapy as first-line treatment from June 1995 through April 2000. Then, patients underwent intensive endoscopic follow-up examinations (+/- endoscopic ultrasonography) with biopsy to evaluate tumor response. Patients with significant improvement of gross lesions that accompanied regression of large cells were followed up without additional treatment. Patients without significant improvement were immediately referred to systemic chemotherapy. RESULTS: Eradication of H pylori was achieved in 15 patients and was accompanied by rapid gross tumor regression and disappearance of large cells in 10. All 10 of these patients with early response had subsequent complete histologic remission of lymphoma. The complete remission rate was 62.5% (95% confidence interval, 35.8% to 89.1%). The response rate was not affected by the tumor grading (proportion of large blast cells within the tumor) but was adversely affected by the depth of tumor invasion. At a median follow-up of 43.5 months (range, 21.1 to 67.4 months), all 10 of these patients remained lymphoma-free. The median duration of complete response was 31.2 months (range, 14.4 to 49.1 months). CONCLUSION: These results suggest that high-grade transformation is not necessarily associated with the loss of H pylori dependence in early-stage MALT lymphomas of the stomach.

Direct comparisons of peripheral T-cell lymphoma with diffuse B-cell lymphoma of comparable histological grades--should peripheral T-cell lymphoma be considered separately?

Peripheral T-cell lymphoma (PTCL) forms a morphologically heterogeneous group of non-Hodgkin's lymphomas (NHL) with distinct immunophenotypes of mature T cells. Progress has been slow in defining specific clinicopathological entities to this particular group of NHL. In order to elucidate the specific characteristics of PTCL, a direct comparison of PTCL with a group of diffuse B-cell lymphomas (DBCL) was performed. Between June 1983 and December 1987, we studied 114 adults with NHL, using a battery of immunophenotyping markers. Adult T-cell leukemia/lymphoma, lymphoblastic lymphoma, mycosis fungoides/Sézary syndrome, follicular lymphoma, well-differentiated lymphocytic lymphoma, and true histiocytic lymphoma were excluded from this study since these are distinct clinicopathologic entities with well-recognized immunophenotypes. Of the remaining 75 patients, 70 who had adequate clinical information were analyzed, and of these, 34 were PTCL and 36 were DBCL. Classified according to the National Cancer Institute (NCI) Working Formulation (WF), 68% of PTCL and 31% of DBCL were high-grade lymphomas. Clinical and laboratory features were similar, except PTCL had a characteristic skin involvement and tended to present in more advanced stages with more constitutional symptoms. Induction chemotherapy was homogeneous in both groups, and complete remission rates were 62% for PTCL and 67% for DBCL. Patients with DBCL had a better overall survival than patients with PTCL, but the survival benefit disappeared after patients were stratified according to intermediate- or high-grade lymphoma. A subgroup of PTCL patients who had received less intensive induction chemotherapy was found to have a very unfavorable outcome. We conclude that (1) PTCL follows the general grading concept proposed in WF classification; (2) within a given intermediate or high grade, PTCL and DBCL respond comparably to treatment; (3) the intensity of induction chemotherapy has a crucial impact on the outcome of PTCL patients; and (4) with a few exceptions, the clinical and laboratory features of PTCL and DBCL are comparable.

Use of retinoic acids in the treatment of peripheral T-cell lymphoma: a pilot study.

PURPOSE: We have systemically analyzed, both in vitro and in vivo, the effect of 13-cis-retinoic acids (RA) on non-Hodgkin's lymphoma (NHL). METHODS: The in vitro growth-inhibitory effect of 13-cis-RA was examined in 11 (T cell, five; B cell, six) lymphoma cell lines by a tetrazolium colorimetric assay. A pilot clinical trial with oral 13-cis-RA 1 mg/kg/d was conducted in a selected group of 18 lymphoma patients, of whom 16 had failed to respond to at least one regimen of intensive chemotherapy. The in vitro and in vivo effects of 13-cis-RA were correlated with immunophenotypes, RA-induced changes of morphology, and patterns of DNA fragmentation of the lymphoma cells. RESULTS: Four of five T-lymphoma cell lines and none of six B-lymphoma cell lines were sensitive (concentration of 50% growth inhibition [IC50] < 1.5 microns) to 13-cis-RA (P = .015). In the clinical trial, five (two Ki-1, one angioinvasive type, one diffuse mixed cell, and one diffuse large cell) complete remissions and one (Ki1) partial remission were observed in 12 patients with peripheral T-cell lymphoma (PTCL), while none of six patients with B-cell lymphoma responded to 13-cis-RA. 13-cis-RA-induced cellular differentiation and apoptosis, as evidenced by the more mature morphology, characteristic nuclear condensation, and DNA ladder pattern signifying internucleosomal fragmentation, were demonstrated in the sensitive cell lines, as well as in the remitting lymphoma tissues. CONCLUSION: The 13-cis-RA appears to be active on lymphomas of T-lineage and their therapeutic indication may be extended to include some subtypes of PTCL. The mechanisms of action are related to differentiation and apoptosis of lymphoma cells. There appears to be no cross-resistance between 13-cis-RA and conventional chemotherapy.

Retinoic acid-induced apoptotic pathway in T-cell lymphoma: Identification of four groups of genes with differential biological functions.

Retinoic acid (RA) has been used to induce the regression of refractory T-cell lymphoma. In vitro and in vivo studies have shown that RA exerts this effect through the induction of apoptosis. This study was designed to investigate the molecular pathway of RA-induced apoptosis in T-lymphoma cell lines.RA-induced apoptosis was verified by morphology, flow cytometry, and DNA ladder analysis. Differential display method using a combination of 12 poly(A)-anchored primers and 20 arbitrary primers was adopted for gene cloning. Total RNAs were extracted from H9 cell line at 0, 6, 12, and 24 hours after All-trans RA (ATRA) treatment and the serial expression patterns of the candidate fragments were recognized. The cloned gene fragments were then analyzed and confirmed by Northern blot analysis on H9 and SR786 cell lines.ATRA-induced apoptosis of T-cell lymphoma was protein synthesis-dependent. The execution or irreversible phase of apoptosis appeared to occur at 6-12 hours of RA treatment. Among the 60,000 arbitrarily displayed bands, 25 of 250 candidate fragments were selected for further cloning and sequencing. A total of 14 clones could be matched to known genes and were categorized into four groups: A) transcription factors: prothymosin, CA150, p78 serine/threonine kinase, IL-1beta-stimulating gene, glucocorticoid receptor, MLN64/CAB1, gastrin-binding protein, and polypeptide from glioblastoma; B) chaperone: 90 kDa heat shock protein; C) ion channel: chloride channel protein 3; and D) cytoskeleton: cytovillin2/ezrin and vimentin. Another two clones of genes were of unrecognized functions. The remaining 11 clones belonged to unmatched or novel genes. The expression of these genes varied, either upregulated or downregulated, in response to ATRA treatment.RA-induced apoptosis may involve a cascade of genes that are related to transcription regulation, stress response, housekeeping, and the execution of apoptosis. The clarification of the RA-induced apoptotic pathway will help us to understand the molecular mechanism of cancer differentiation agents.

Report of the Workshop on Nasal and Related Extranodal Angiocentric T/Natural Killer Cell Lymphomas. Definitions, differential diagnosis, and epidemiology.

A workshop jointly sponsored by the University of Hong Kong and the Society for Hematopathology explored the definition, differential diagnosis, and epidemiology of angiocentric lymphomas presenting in the nose and other extranodal sites. The participants concluded that nasal T/natural killer (NK) cell lymphoma is a distinct clinicopathologic entity highly associated with Epstein-Barr virus (EBV). In situ hybridization for EBV an be very valuable in early diagnosis, especially if tissue is sparse. The cytologic spectrum is broad, ranging from small or medium-sized cells to large transformed cells. Histologic progression often occurs with time. Necrosis is nearly always present, and angioinvasion by tumor cells is seen in most cases. Nasal T/NK cell lymphoma has a characteristic immunophenotype: CD2-positive, CD56-positive, but usually negative for surface CD3. Cytoplasmic CD3 can be detected in paraffin sections. Clonal T-cell receptor gene rearrangement is not found. Tumors with an identical phenotype and genotype occur in other extranodal sites, most commonly in the skin, subcutis, and gastrointestinal tract, and should be referred to as nasal-type T/NK cell lymphomas. The differential diagnosis includes lymphomatoid granulomatosis, blastic or monomorphic NK cell lymphoma/leukemia, CD56-positive peripheral T-cell lymphoma, and enteropathy-associated T-cell lymphoma.

Epstein-Barr virus-associated intravascular lymphomatosis within Kaposi's sarcoma in an AIDS patient.

Intravascular lymphomatosis (IL) is an unusual neoplasm characterized by multifocal proliferation of lymphoma cells exclusively within the blood vessels. We report here a patient with acquired immunodeficiency syndrome (AIDS) and disseminated Kaposi's sarcoma. A 233-bp amplification product of HHV-8 was detected in the DNA extracted from specimens of Kaposi's sarcoma at different sites by polymerase chain reaction (PCR). At autopsy, the vessels within the Kaposi's sarcoma were dilated and filled with atypical large mononuclear cells. No such feature was seen in the vessels of non-Kaposi's sarcomatous regions. Immunohistochemically, the spindle cells of Kaposi's sarcoma were positive for CD31 (endothelial cell marker). The intravascular tumor cells were positive for CD45 (leukocyte common antigen) but negative for others, including chloroacetate esterase, CD45-RO (UCHL-1, Pan-T), CD3, CD43, CD20 (L26, Pan-B), CD30 (Ki-1), immunoglobulin heavy chains and light chains, CD56 (natural killer cell antigen), and CD31. Monoclonal rearrangement of immunoglobulin heavy chain gene was detected in the DNA extracts from fresh tissue of Kaposi's sarcoma by PCR, which indicated that the lymphoma cells within the Kaposi's sarcoma were of monoclonal B cell origin. In situ hybridization revealed that EBER-1 transcripts were present in the lymphoma cells of IL but not in the spindle cells of Kaposi's sarcoma. To the authors' best knowledge, this is the first instance of IL in an AIDS patient with direct evidence of EBV association.

Lac/Tet dual-inducible system functions in mammalian cell lines.

The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels. In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms). The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell. The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes. In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.

Experience of radiotherapy in lethal midline granuloma with special emphasis on centrofacial T-cell lymphoma: a retrospective analysis covering a 34-year period.

Lethal midline granuloma (LMG) is characterized by progressive ulceration and destruction of the midfacial tissue. It occurs more frequently in Oriental than in Western populations. Because of the progress in clinical pathology and immunohistochemistry, most cases have been proven to be malignant lymphomas, especially of T-cell lineage. We describe 92 cases of lethal midline granuloma or centrofacial malignant lymphoma in the period 1959-1993. All received complete courses of radiotherapy. Twenty of them also received combination chemotherapy. Thirty-six cases had specimens available for immunohistochemical study; 25 (69%) of these had a T-cell phenotype, and 6 (17%) were of B-cell lineage. The dose to the nasal region was in the range of 3000-7500 cGy in 11-58 days, and to the neck 3000-6400 cGy in 11-48 days. The overall survival rate for the LMGs was 59.5% at 5 years and 56.2% at 10 years (Kaplan-Meier). Combined chemotherapy seemed not to improve the overall survival in this study (p = 0.63), but the patient number was too small to make a firm conclusion. Based on the results of this study, we recommend a dose of 4500-5000 cGy to the midfacial region, since a higher dosage did not improve the treatment results (p = 0.88). Irradiation has a definite role in good locoregional control of this disease. The recent clarification of the disease nature and the recognition of the background clinicopathological features should provide valuable information for future patient management and prospective studies.

Immunoglobulin and T-cell receptor gene rearrangements in acute lymphoblastic leukemia--a higher incidence of double rearrangements in patients with myeloid antigen expression.

Among 160 patients who were diagnosed as having acute lymphoblastic leukemia (ALL) by French American British (FAB) criteria, 32 patients (20%) expressed myeloid-associated antigens on leukemic blasts (My+ALL). Correlation of immunophenotype with rearrangement of immunoglobulin (Ig) heavy chain and T-cell receptor (TCR) beta chain genes was performed on 73 of these patients (21 were My+ALL). Rearrangements of both Ig and TCR genes (double rearrangements) were detected in 24 patients, including three (19%) of 16 T-lineage ALL. 17 (33%) of 52 B-lineage ALL, and four of five ALL expressing both B and T-cell surface markers. Also a higher incidence of double rearrangements in My+ALL was found as compared with My-ALL (43% vs 29%). This difference was more evident when only B-lineage ALL was considered (50% in My+ patients vs 24% in My- patients). However the difference is not statistically significant yet possibly due to the small number of patients in the study. Further studies on more patients are needed to confirm this. In My-B-lineage ALL, rearrangements of TCR beta chain gene were restricted to certain subgroups (Groups III & IV) of patients who expressed CD10 surface antigens but lacked cytoplasmic Ig. In My+ B-lineage ALL, rearrangements of TCR beta chain gene could be found in various subgroups studied (Groups II through V). Cross-lineage gene rearrangement in My+ALL may involve mechanisms different from those in My-ALL.

A subset of acute nonlymphocytic leukemia with expression of surface antigen CD7--morphologic, cytochemical, immunocytochemical and T cell receptor gene analysis on 13 patients.

An increasing number of acute leukemias coexpressed markers normally believed to be restricted to a single lineage have been found recently. This special subgroup of leukemias have drawn a lot of attention because of their biologic and clinical significance. In a study of 100 consecutive de novo ANLL patients diagnosed by FAB criteria, T-cell antigen CD7 was identified on the leukemic blasts of 13 patients, ten of whom had M1 subtype of leukemia, myeloblastic leukemia without maturation. All the patients showed positive staining with myeloperoxidase and expressed myeloid markers CD13 and/or CD33, but lacked CD11b, a marker of more mature myeloid cells. Combined staining with myeloperoxidase and CD7 of the cells from four patients revealed coexpression of both markers on the same cells. None of the patients expressed the two other T-cell antigens CD2 or CD5. All ten patients who had DNA analysis showed germline configuration of TCR beta and gamma chain genes. One patient had chromosomal translocation involving 11q23, t(11; 19) (q23; p13), which is the site frequently associated with both myeloid and lymphoid malignancies. The clinical implications of this subgroup of patients need further study on more patients, and need longer follow-up.

Detection of bcl-2 and p53 in thymoma: expression of bcl-2 as a reliable marker of tumor aggressiveness.

The distinction between noninvasive and invasive or malignant thymoma has been severely compromised by a lack of objective morphological criteria. A reliable marker of tumor aggressiveness is, therefore, mandatory for predicting the tumor behavior. Forty thymic epithelial tumors, including 5 noninvasive thymomas, 18 invasive thymomas, and 17 thymic carcinomas (Rosai's classification) were investigated for expression of bcl-2 and p53 proteins by immunohistochemistry. The thymic epithelial cells showed positive immunostain for bcl-2 in 0, 7, and 16 of these categories, respectively. Thymic carcinomas had a significantly higher proportion of bcl-2 expression than thymomas (P < .0001). A significantly higher expression of bcl-2 protein was also shown in thymoma-associated myasthenia gravis (P < .05). However, p53 showed no correlation with the histological subtypes nor clinical aggressiveness. Bcl-2 expression appeared to be positively correlated with p53 immunoreactivity, but this result was not statistically significant (P = .07). In conclusion, these data indicate that bcl-2 expression correlates with aggressiveness in thymic epithelial neoplasms.