Competitive repopulation assay of two gene-marked cord blood units in NOD/SCID/gammac(null) mice.

In multiunit cord blood transplantation, hematopoietic stem cells from each unrelated cord blood (UCB) unit competitively reconstitute the hematopoietic system in a recipient. To evaluate the fate of the progeny of each UCB unit and to determine the effects of graft-versus-graft reaction, we established a novel competitive repopulation assay using NOD/SCID/gammac(null) mice in which human T lymphocytes develop from CD34+ cells. CD34+ cells from each UCB unit were labeled with recombinant lentivirus vectors carrying genes encoding either enhanced green fluorescent protein (EGFP) or enhanced yellow fluorescent protein (EYFP). Hematopoietic chimerism composed of both EGFP+ and EYFP+ cells was stably maintained up to 6 months after transplantation with purified CD34+ cells; the ratio of EGFP+ to EYFP+ cells in peripheral blood and bone marrow posttransplantation was equivalent to the ratio of these cells at transplantation. However, when mononuclear cells from two UCB units were cotransplanted with CD34+ cells, engraftment was highly competitive, with cells from only one or the other of the two UCB units surviving. Further subfractionations of mononuclear cells indicate that the skewed chimerism that is often observed in clinical multiunit cord blood transplantation may be mediated by the cooperation of both CD4+ and CD8+ T cells. The assay established here will be a useful tool for analyzing hematopoietic reconstitution in clinical multiunit cord blood transplantation.     

Prognostic analysis and a new risk model for Hodgkin lymphoma in Japan

The Japan Clinical Oncology Group conducted two multicenter phase II trials in 200 patients with advanced Hodgkin lymphoma (HL) in the 1990s. Among 181 patients whose histopathological specimens were available and reviewed by 6 hematopathologists, 167 (92.3%) were diagnosed with HL. Five-year overall survival (OS) among these 167 patients was 88.3%, including 89.2% among nodular sclerosis and 82.2% among mixed cellularity cases. International prognostic score was not closely associated with OS. Seven unfavorable prognostic factors for OS on univariate analysis were male, B symptoms, clinical stage of III or IV, elevated serum LDH, elevated alkaline phosphatase, elevated β2-microglobulin, and pathological subtype (mixed cellularity and lymphocyte depletion). On multivariate analysis, male [HR 3.30 (95% CI 1.15–9.52, p = 0.027)] and elevated serum LDH [HR 2.41 (95% CI 1.07–5.43, p = 0.034)] were independent factors for OS. Based on these prognostic factors, the 5-year OS was 95.7% in the low-risk group (no adverse factor), 87.9% in the intermediate-risk group (1 adverse factor) and 73.3% in the high-risk group (2 adverse factors). This simple prognostic model for HL warrants further validation studies.     

Remission of lymphoma after withdrawal of methotrexate in rheumatoid arthritis: relationship with type of latent Epstein-Barr virus infection

Rheumatoid arthritis (RA) is associated with an increased risk of developing lymphoma. Although the pathogenesis is still unclear, the increased risk appears to be related to the high inflammatory activity of RA, immunosuppressive agents, or Epstein-Barr virus (EBV) infection. We investigated the relationship between EBV latent infection and methotrexate (MTX)-associated lymphoma in RA patients. Nine patients were diagnosed with non-Hodgkin's lymphoma (NHL) during MTX treatment for RA in a multicenter study. The pathologic findings were consistent with diffuse large B-cell lymphoma in 8 patients and peripheral T-cell lymphoma, unspecified in 1. EBV infection was detected in 3 patients by in situ hybridization. Among all 9 patients who were initially treated by MTX withdrawal alone, 2 obtained spontaneous complete response (CR), 1 had partial response, 2 had stable disease (SD), and 4 had progressive disease. Both patients who had a CR and 1 who had SD were positive for EBV. Further examination of the latent EBV infection patterns revealed that 2 patients who obtained a CR had latency Type III, and the other with SD had latency Type II. These results demonstrate that immunodeficiency caused by MTX treatment is associated with the development of EBV-related NHL in RA patients. In patients who were treated by MTX for RA and developed NHL, remission can be observed following MTX withdrawal especially in NHL with latency Type III EBV infection. The analysis of EBV infection, including the latency types, is useful to decide the optimum therapeutic strategy.     

Promoter hypomethylation of protease-activated receptor 2 associated with carcinogenesis in the stomach

BACKGROUND AND AIM: Trypsin acting at protease-activated receptor 2 (PAR2) contributes to a progression of malignant tumors. An abnormal DNA methylation has been recognized as an important molecular mechanism for the genesis of various types of cancers. We attempted to clarify the relationship between the promoter methylation of PAR2 and gastric cancer. METHOD: We estimated the methylation of the PAR2 promoter in both antral non-cancerous mucosa and cancer lesions in 94 patients with gastric cancer. We employed a methylation-specific PCR method. RESULTS: Regarding the methylation ratio (MR) of antral-non-cancerous mucosa, no significant difference was despite among gender, age and Helicobacter pylori infection status, whereas MR increased rising inflammation scores. The MR of cancer lesions was significantly lower than that of antral non-cancerous mucosa. This finding was not dependent on tumor staging, but also histological classification. In venous invasion, lymph node metastasis, or peritoneal dissemination negative cases, this significant lower MR was also seen. CONCLUSION: The promoter methylation of PAR2 seems to be increased with a progression of chronic inflammation and has an inhibitory effect on carcinogenesis of the stomach.     

Effect of polymorphisms of IL-1β and TNF-α genes on CpG island hyper methylation (CIHM) in the nonneoplastic gastric mucosa

CpG island hyper methylation (CIHM) is one of the major events in gastric carcinogenesis. To evaluate the influence of host genetic factors in CIHM related carcinogenesis, we investigated the association between common polymorphisms in IL-1β and TNF-α genes, with CIHM status in the nonneoplastic gastric mucosa. Polymorphisms in the IL-1β gene (-31T>C and -511C>T) and the TNF-α gene (-857C>T) were genotyped in 385 cancer-free subjects. CIHM of four candidate genes: p16 (INK4a), p14 (ARF), E-cadherin (CDH1), and death-associated protein kinase (DAP-kinase), were determined by methylation-specific-polymerase chain reaction (MSP). CIHM high was defined as two or more CpG islands methylated. CIHM of all four genes and CIHM high were significantly associated with Helicobacter pylori infection status. In over all, significant marginal association was found between IL-1β-511 TT genotype and reduced susceptibility to CIHM of DAP-kinase (adjusted OR = 0.48, 95% CI = 0.29-0.78) and CIHM high (adjusted OR = 0.53, 95% CI = 0.32-0.86). This association was more enhanced in subjects 65 yr or younger age. We also found positive association between TNF-α-857T carrier and increased susceptibility to CIHM of CDH (adjusted OR = 1.78, 95% CI = 1.01-3.16), and CIHM high (adjusted OR = 1.86, 95% CI = 1.04-3.33) in the same generation. The mean number of CIHM was lower in subjects with IL-1β-511TT genotype, while the mean number was higher in subjects with TNF-α-857 T carrier especially in subjects 65 yr and younger patients. IL-1β-511 TT genotype is associated with reduced susceptibility to CIHM especially in younger generation. Furthermore, the TNF-α-857T carrier is associated with increased susceptibility of CIHM in the same generation.