Chemiluminescence Immunoassay to Detect Anti-HTLV-I Antibodies

A fully automated chemiluminescence immunoassay was developed for the detection of antibodies to HTLV-I. We used partially purified viral antigens coated on small polystyrene beads together with acridinium ester-labeled anti-human immunoglobulin G mouse immunoglobulin G in this method. A good agreement was observed between our proposed method, the indirect immunofluorescence assay, the particle-agglutination test and the enzyme immunoassay. This new method, which is simple, sensitive, specific and rapid, should be useful for mass screening of anti-HTLV-I antibodies.     

Effect of Pseudomonas aeruginosa exotoxin A on endotoxin-induced tumour necrosis factor production in murine lung

The ability of several Pseudomonas aeruginosa exo-enzymes, including exotoxin A (ETA), to induce inflammation and their influence on endotoxin-induced tumour necrosis factor (TNF) production in murine lung were evaluated. Intratracheal administration of lipopolysaccharide (LPS; 0.1-10 microg/mouse), 2(-1) LD50 of P. aeruginosa alkaline protease (7.5 microg/mouse) and elastase (1.2 microg/mouse) elevated total cell number and the percentage of neutrophils in broncho-alveolar lavage fluid (BALF), whereas ETA (0.1 microg/mouse) did not. LPS induced TNF production in BALF in a dose-dependent manner, whereas the P. aeruginosa exo-enzymes did not. When ETA was inoculated into the respiratory tract before LPS, production of TNF in BALF was significantly suppressed in a dose-dependent manner. ETA also suppressed TNF production by alveolar macrophages (AMs) stimulated with LPS in vitro. Flow cytometric analysis showed that ETA markedly reduced the expression of CD14 and CD11c/CD18 on the surface of AMs. ETA also depressed partially the expression of TNF-alpha mRNA in AMs. These findings suggest that ETA regulates TNF production in murine lung by suppressing LPS receptor expression, mRNA expression and protein synthesis and/or secretion of TNF.     

Flow-cytometry detection of minimal DNA aneuploidy in mature lymphoid leukemias - comparison with metaphase and interphase cytogenetics

The relative DNA content of peripheral blood cells from 79 cases with lymphoid leukemias was analyzed by a dual-parameter flow cytometric analysis. The leukemia samples corresponded to: chronic lymphocytic leukemia (CLL) 40, CLL with more than 10% prolymphocytes (CLL/PL) 12, CLL mixed 9, prolymphocytic leukemia (PLL) 5, and B-cell lymphoma in leukemic phase 13. DNA aneuploidy was found overall in 26 (32.9%) of the cases and these corresponded to: 7 (17.5%) with CLL, 7 (58.3%) with CLL/PL, 4 (44.4%) with CLL mixed, 2 (40%) with PLL and 6 (46.2%) with B-cell lymphoma. There was a good correlation between DNA content and cytogenetics/fluorescent in situ hybridization in all but 2 cases as follows: 6 of 7 cases with diploid DNA had normal karyotype and only one had trisomy 12: 4 of 6 cases with hyperdiploid DNA had trisomy 12, one had tetraploidy and only one had a normal karyotype. Two cases were hypodiploid both by DNA and cytogenetic analysis. Our findings demonstrate a higher incidence of DNA aneuploidy in B-cell lymphoma in leukemic phase, PLL, and atypical CLL in comparison with typical CLL and a good correlation with cytogenetics. We conclude that flow cytometric DNA analysis represents a useful, sensitive, and rapid method to detect and monitor minimal changes of DNA content in leukemic lymphocytes without the need of short-term cultures.     

High establishment efficiency of lymph node stromal cells which spontaneously produce multiple cytokines derived from adult T-cell leukemia/lymphoma patients

Stromal cells isolated from lymph nodes of adult T-cell leukemia/lymphoma (ATL) patients were cultured. Such lymph node stromal cells (LNSC) could be maintained for more than one year, whereas LNSC from other lymphoproliferative disorders ceased to proliferate within months. The rate of human T lymphotropic virus type I (HTLV-I) integration in these LNSC was examined by nested polymerase chain reaction (PCR) and estimated to be about 1 genome per 100 cells. These LNSC showed the same combination of cytokine production irrespective of the patient origin, granulocyte-macrophage (GM)-CSF, G-CSF, interleukin (IL)-1 beta, IL-6, interferon (IFN)-gamma and IL-8, being positive but not M-CSF, IL-1 alpha, IFN-alpha, tumor necrosis factor (TNF)-alpha, IL-2, LD78 and the IL-1 receptor antagonist (IL-1ra). The results show that LNSC from ATL patients have pronounced proliferation activity and constitutively secrete various cytokines. They therefore provide useful models for studying the microenvironment of lymph nodes in vitro, and especially the growth mechanism of ATL cells.     

Suppressor of cytokine signal 3 and IL28 genetic variation predict the viral response to peginterferon and ribavirin

Aim: The aim of this study was to investigate the relationship among the expression of suppressor of cytokine signaling 3 (SOCS 3) in the liver, the SNPs in the IL28B locus, and the outcome of interferon therapy. Methods: Prior to interferon treatment, we immunostained 67 liver specimens from chronic hepatitis C (CHC) patients who were receiving peginterferon alpha‐2b/ribavirin therapy for suppressor of cytokine signaling 3 (SOCS3), and compared the expression of SOCS3, IL28 polymorphisms and other clinical factors between the patients and compared their eventual outcomes. Results: Significant differences between the low SOCS3 group and high SOCS3 group were found in age, as well as in the platelet, transaminase, gamma‐glutamyl transpeptidase levels. The incidence of high SOCS3 was not significantly different between subjects with the TT genotype and the TG genotype (TT : TG = 71%:29%, P = 0.250). In a multivariate analysis, age (≥65 years old) (odds ratio 0.221 [0.120–0.966], P = 0.045), IL28B gene (genotype TT) (odds ratio 5.422 [1.254–23.617], P = 0.024) and SOCS3 (high) (odds ratio 0.308 [0.104–0.948], P = 0.040) were significant predictors of the interferon response. In patients with the TT genotype, those with low SOCS3 immunostaining showed a high sustained virological response (69%), while the sustained virological rate was low (27%) in the patients with high SOCS3 immunostaining. Conclusions: Using a combination of the SOCS3 immunostained area in the liver and the expression of IL28B single nucleotide polymorphisms might be a useful predictor of hepatitis C virus clearance by interferon therapy.     

Successful treatment of a chronic-phase T-315I-mutated chronic myelogenous leukemia patient with a combination of imatinib and interferon-alfa

The T315I BCR-ABL mutation in chronic myelogenous leukemia (CML) patients is responsible for up to 20% of all clinically observed resistance. This mutation confers resistance not only to imatinib, but also to second-generation BCR-ABL tyrosine kinases, such as nilotinib and dasatinib. A number of strategies have been implemented to overcome this resistance, but allogeneic stem cell transplantation remains the only established therapeutic option for a cure. A 61-year-old male was diagnosed with Philadelphia chromosome-positive chronic-phase CML in 2002. He was initially treated with imatinib and complete cytogenetic response (CCyR) was achieved 12 months later. However, after 18 months, a loss of CCyR was observed and a molecular study at 24 months revealed a T315I mutation of the BCR-ABL gene. At 30 months, imatinib/interferon-alfa (IFNα) combination therapy was initiated in an effort to overcome the resistance. Thirty months later, he re-achieved CCyR, and the T315I BCR-ABL mutation disappeared at 51 months. To our knowledge, this is the first case report showing the effectiveness of imatinib/IFNα combination therapy for CML patients bearing the T315I BCR-ABL mutation.