ALBERT

Description: Endothelial cells are linked to each other through intercellular junctional complexes that regulate the barrier and fence function of the vascular wall. The nature of these intercellular contacts varies with the need for permeability: For example, in brain the impervious blood-brain barrier is maintained by “tight” contacts between endothelial cells. By contrast, in high endothelial venules (HEVs), where lymphocytes continuously exit the bloodstream, the contacts are generally leaky. The precise molecular components that define the type of junction remain to be characterized. An immunoglobulin superfamily molecule named JAM-2, specifically expressed in lymphatic endothelial cells and HEVs, was recently identified. JAM-3 was cloned and characterized in the current study, and JAM-1, -2, and -3 were shown to form a novel protein family belonging to the larger cortical thymocyte Xenopus (CTX) molecular family. Using antibodies specific for each of the 3 family members, their specific participation in different types of cell-cell contact in vivo and their specific and differential localization in lateral contacts or tight junctions were demonstrated. Furthermore, it was shown that JAM-1 and JAM-2 differentially regulate paracellular permeability, suggesting that the presence of JAM-1, -2, or -3 in vascular junctions may play a role in regulating vascular function in vivo.

Description: STI571 is a highly effective drug for the therapy of CML, but there is still drug resistance, especially in blast crisis. To study the possible mechanisms of resistance to STI571, we established the BCR/ABL+ cell line with resistance to STI571 (K562-R) in vitro by culturing a wild-type K562 cells (K562-W) in gradually increased concentrations of STI571 over a period of months. Trypan blue staining, MTT assay and Hoechst 33342 staining confirmed that K562-R can live steadily at 0.5umol/L STI571. Furthermore MDR-1 expression assay, sequence analysis, fluorescence in situ hybridization(FISH) and cDNA array were used to study the potential mechanisms of acquired resistance. The MDR-1 expression percentages of K562-W and K562-R with FASC analysis were 2.68% and 1.39% respectively. No point mutant in the BCR/ABL ATP-binding site was detected and the copies of BCR/ABL fusion gene were found increased in K562-R by FISH analysis. By a expression profile of cDNA microarray, 327 genes’ expression were found down-regulating including one of homo sapiens protein tyrosine phosphatase genes(PTPRF) and 335 genes up-regulating including homo sapiens hematopoietic cell-specific Lyn substrate 1 gene(HCLS1). Our studies proved the possible mechanism of K562-R resistance involved amplification of BCR/ABL fusion gene and increase of phosphorylation activity in this cell line.

Description: BCL10 is directly involved in t(1;14)(p22;q32) of mucosa-associated lymphoid tissue (MALT) lymphoma. Wild-type BCL10 promoted apoptosis and suppressed malignant transformation in vitro, whereas truncated mutants lost the pro-apoptotic activity and exhibited gain of function enhancement of transformation. We studied 220 lymphomas for genomic BCL10 mutation by polymerase chain reaction–single-strand conformational polymorphism and DNA sequencing. Nineteen mutations were found in 13 lymphoma specimens, as follows: 8 of 120 (6.7%) mucosa-associated lymphoid tissue (MALT) lymphomas, 4 of 42 (9.5%) follicular lymphomas, and 1 of 23 (4.3%) diffuse large B-cell lymphomas. No mutations were found in 14 mantle cell lymphomas or 21 T-cell lymphomas. High-grade MALT lymphoma tended to show a slightly higher mutation frequency (2 of 25, 8%) than low-grade MALT tumor (6 of 95, 6.3%). Among low-grade gastric MALT lymphoma, mutations were found in 3 of 11 tumors that did not respond to Helicobacter pylori eradication therapy, but none were found in 22 tumors that regressed completely after H pylori eradication. All 14 potentially pathogenic mutations were distributed in the carboxyl terminal domain of BCL10. Deletion accounted for 10 of these mutations; 10 of 14 mutations caused truncated forms of BCL10. Western blot analysis of a mutant case confirmed the presence of truncated BCL10 products of anticipated size. Our results suggest that BCL10 mutation may play a pathogenic role in B-cell lymphoma development, particularly in aggressive and antibiotic unresponsive MALT lymphomas, and may further implicate the biologic importance of the carboxyl terminal of the molecule.

Description: Hematopoietic cancer is thought to arise from small populations of immortalized cancer stem cells that give rise to phenotypically diverse cells with less proliferative capacity. Cancer stem cell immortalization genes represent attractive drug targets for treating human cancer. We have found that immortalized cell lines can be generated in vitro following infection of mouse bone marrow cells with replication defective murine stem cell virus (MSCV). Immortalized myeloid progenitors can be selected in the presence of SCF plus IL-3 while immortalized multi-potent early hematopoietic progenitor cells can be selected with SCF and Flt3 ligand. So far, more than 80 such lines have been generated and they exhibit many properties attributed to cancer stem cells; they undergo unlimited self-renewal yet are able to give rise to phenotypically diverse cells with less proliferative capacity and in some cases they are leukemogenic in transplanted hosts. Immortalization often results from the insertional mutagenesis of human leukemia gene orthologues or genes that regulate them. Insertional mutagenesis by MSCV thus produces cells that share many properties attributed to cancer stem cells and may mark a subset of genes important for the immortalization of leukemic stem cells.

Description: RIT produces high rates of durable complete responses in “low grade” NHL. Clinical studies have thus far been limited to a single infusion of a radiolabeled anti-CD20 mAb (131I tositumomab or 90Y ibritumomab tiuxetan) in patients with 〈 25% bone marrow involvement. In this study we have tested the safety and efficacy of 4 weekly infusions of Rituximab followed by 2 fractions of 131I labelled rituximab given 8 weeks apart in relapsed "low grade" NHL and have included patients with higher levels than 25% bone marrow infiltration with lymphoma. Whole body dose (WBD) calculations have been used to allow the total dose given in 2 fractions to be increased in cohorts of 30cGy, 60cGy, 90cGy and 120cGy. A unique anti-rituximab idiotype mAb has been generated enabling serial analysis of serum rituximab concentrations (Cragg et al Blood 2004 Jun 22 Epub). This tool has allowed us to accurately quantify the serum levels of rituximab during the entire treatment schedule. We have also analysed the impact made by induction rituximab, the pre-dose of cold antibody and effects of fractionated RIT on the clearance and biodistribution of the 131I labeled rituximab. Sequential pharmacokinetic analyses have identified wide variation in the effective half-life of 131I-rituximab not only between patients but also within the same patient over the course of the treatment protocol. We found that the mean effective half-life of 131I rituximab increased from 43 hours prior to induction rituximab to approximately 106 hours prior to the second fraction of 131I-rituximab. A strong inverse correlation was found between the patients’ disease burden and the clearance of rituximab, both in the same patient as the tumor burden decreased and between different patients. We have demonstrated that higher cumulative WBD doses can be safely delivered by RIT dose fractionation than can be given with a single dose of anti-CD20, with no grade 3–4 myelotoxicity at the 90 cGy dose level. All 12 patients in the first 3 dose cohorts experienced responses and response durations equal to, or in the majority of cases, superior to that seen with their previous chemotherapy regimen. Data on all 16 patients including 120 cGy dose level will be available at the time of presentation. In conclusion, this fractionated RIT protocol is feasible, efficacious and enables larger WBD doses to be safely delivered than has previously been achieved with non-myeloablative RIT.

Description: The development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma is a multistep process and can be clinico-pathologically divided into Helicobacter pylori-associated gastritis, low-grade tumors, and high-grade tumors. The molecular events underlying this progression are largely unknown. However, identification of the genes involved in MALT lymphoma-specific t(11;18)(q21;q21) and t(1;14)(p22;q32) has provided fresh insights into the pathogenesis of this disease. T(11;18)(q21;q21) results in a chimeric transcript between the API2 and theMALT1 genes, whereas t(1;14) (p22;q32) causes aberrant nuclear BCL10 expression. Significantly, nuclear BCL10 expression also occurs frequently in MALT lymphomas without t(1;14)(p22;q32), suggesting an important role for BCL10 in lymphoma development. Thirty-three cases of H pylori gastritis, 72 MALT lymphomas, and 11 mucosal diffuse large B-cell lymphomas (DLBCL) were screened for t(11;18)(q21;q21) by reverse transcription–polymerase chain reaction followed by sequencing. BCL10 expression in lymphoma cases was examined by immunohistochemistry. The API2–MALT1 fusion transcript was not detected in H pylorigastritis and mucosal DLBCL but was found in 25 of 72 (35%) MALT lymphomas of various sites. Nuclear BCL10 expression was seen in 28 of 53 (53%) of MALT lymphomas. Of the gastric cases, the largest group studied, the frequency of both t(11;18)(q21;q21) and nuclear BCL10 expression was significantly higher in tumors that showed dissemination to local lymph nodes or distal sites (14 of 18 = 78% and 14 of 15 = 93%, respectively) than those confined to the stomach (3 of 29 = 10% and 10 of 26 = 38%). Furthermore, t(11;18)(q21;q21) closely correlated with BCL10 nuclear expression. These results indicate that both t(11;18)(q21;q21) and BCL10 nuclear expression are associated with advanced MALT lymphoma and that their oncogenic activities may be related to each other.

Description: Human Herpes virus 8 (HHV8) associated multicentric Castleman’s disease (MCD) is an unusual multifocal lymphoid hyperplasia induced by HHV8 infected B cells and associated with a characteristic systemic syndrome attributed to raised levels of IL-6. Most cases develop on a background of immunosuppression, often as a result of human immunodeficiency virus (HIV) infection. Despite the haematological problems at presentation and the difficulties in initial diagnosis, the bone marrow appearances of MCD have not been so far described. In this study we examined the pathology of bone marrow in 13 patients with MCD, 11 of whom had HIV infection, with a view to identifying features that may be helpful in early diagnosis. Patients typically presented with fever, lymphadenopathy, hepatosplenomegaly and cytopaenias. Bone marrow aspirates showed mild to moderate trilineage dysplasia, a mild plasmacytosis, and a mild eosinophilia similar to that seen in HIV infected patients without MCD. Bone marrow biopsies showed hypercellularity, architectural disorganisation, variably prominent dysplasia especially in megakaryocytes, mild eosinophilia, and a polytypic plasmacytosis representing 5–20% of all cells. Interestingly, two cases showed marked megakaryocytic and granulocytic hyperplasia with reticulin fibrosis, similar to the effects of IL-6 on the marrow in experimental systems. Importantly, in 3 cases there were small lymphoid follicles typical of MCD in diagnostic nodal specimens. Depleted germinal centres were surrounded by mantle zones containing scattered large plasmablasts which expressed HHV8 latent nuclear antigen (LNA) and showed lambda immunoglobulin light chain restriction. Furthermore, varying numbers of dispersed interstitial HHV8-LNA positive plasmablasts were present in 11/13 cases. Double immunohistochemical staining confirmed the B cell phenotype of these plasmablasts. The presence of these cells was highly specific for MCD as rare single HHV8 positive cells were identified in only 4 of 66 control bone marrow biopsies from HIV positive patients. Each of these 4 patients had Kaposi’s sarcoma and 1 also had a primary effusion lymphoma. HHV8 positive cells were not identified in bone marrow biopsies from 23 other HIV positive patients with lymphoma. These results suggest that the presence of HHV8 positive plasmablasts in bone marrow biopsies, within characteristic lymphoid follicles and/or the interstitium, is highly specific and sensitive for MCD. As the examination of bone marrow is the first diagnostic test performed in virtually all MCD patients, the features described in this study should greatly enhance the chances of early diagnosis by either providing the tissue diagnosis or prompting a lymph node biopsy and further investigations. Furthermore, the HHV8 positive cells within the bone marrow may play an important pathogenetic role in the haematological disturbances typically seen in MCD.

Description: The prevalence of subgroups of myelodysplastic syndrome (MDS) was determined in an unselected series of 92 patients presenting at Shanghai hospitals over a 10 month period. Diagnosis was established in a single laboratory, analyzing morphologic, immunophenotypic, fluorescence in situ hybridization (FISH) and cytogenetic data, according to the World Health Organization (WHO) revised classification. The frequencies of the major MDS subgroups were: refractory anemia (RA) 9.7%, refractory anemia with ringed sideroblasts (RARS) 1%, refractory anemia with excess blasts (RAEB) 15.2%, refractory cytopenia with multilineage dysplasia (RCMD) 52.6%, and MDS-unclassifiable (MDS-u) 7.6%. The subgroup, myelodysplastic/myeloproliferative diseases, unclassifiable (MDS/MPD-u) (9.7%) was also included for comparison with other studies. The median age at diagnosis for all cases was 57 yr. Males slightly predominated (53%). The overall incidence of clonal cytogenetic abnormalities was 24.7%. The most frequently encountered lesions were trisomy 8, 20q-, 5q- and 7q-. The prevalence of clonal cytogenetic abnormalities in each subgroup differed significantly from none in RA to 31% in RCMD. Deletions involving 5q- and 7q- were found predominantly in RAEB and RCMD subgroups. The incidence of MDS in China has been reported to be less than that in the West. However, information on the frequency of different subgroups of MDS in Shanghai previously has not been available. These cases presented at 24 hospitals serving a population of approximately 18 million. These results suggest major differences in the presentation and frequency of different subgroups of MDS between China and the West.

Description: The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive condition manifesting as variable degrees of immunodeficiency and thrombocytopenia. The strong clinical improvement in platelet count seen in WAS patients after splenectomy suggests that increased platelet consumption is a major contributor to the thrombocytopenia. Here we show that splenectomy improves but does not normalize platelet count in a murine (C57Bl/6) model of WAS, suggesting (a) that murine WAS parallels clinical WAS in this regard, and (b) that either increased extrasplenic consumption or diminished platelet production (or both) contributes to the thrombocytopenia. We quantified platelet turnover by labeling WAS and WT platelets with CMFDA and following their disappearance after injection into WT recipients. Platelet loss rates in this model fit best to a simple exponential decay curve. We found that WAS platelets turn over significantly faster than WT (survival times: 56 hr (WT) vs 40 hr (WAS), p

Description: Introduction The extensive diversity of the mature T-cell receptor(TCR) is determined primarily by the complementarity-determining regions (CDR3) of the TCR. The CDR3 of both TCRα and TCRβ genes is generated by extensive rearrangement and fusion between the V,D,and J segments and by random insertion and deletion of junctional nucleotides, which yields final products that are quite heterogeneous in size. As a result of these gene rearrangements, each T cell has a unique TCR and the diversity of the T-cell repertoire at any specific time can be characterized by the examination of CDR3 within that population. Using CDR3 spectratying technique, normal individuals demonstrate a highly diverse and polyclonal The aim of our study was to evaluate to investigate restricted expansion of TCR Vβ gene repertoire and the reconstitution of T cell receptor repertoire following allogeneic hematopoietic stem cell transplantation. Methods Patients Ten patients(9 males, 1 females; median age 31 years,range18–45) with 6 chronic myeloid leukemia-chronic phase and 4 cases of acute myelogenous lenkemia(CR1) who underwent HLA-matching sibling or unrelated BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department between July 1999 and May 2000 were considered evaluable restricted expansion of TCR Vβ gene repertoire, the reconstitution of T cell receptor repertoire and oligoclonal T Cell Expansion in Chronic Graft-Versus-Host Disease. RT-PCR and Genes scan analysis (CDR 3 length analysis). Results Only 2-18Vβ genes were found in samples from these ten patients within one year, and there are different distribution in different patients. TCR repertoire complexity was abnormal in all patients, parts of the genes were expansion and part of them were suppressed. Samples from 9 patients with GVHD show V β3 in 7 cases, V β 8 and V β 23 in 6 patients. The results of genescan show that the PCR production of peripheral blood samples from these patients disply oligoclonal. Only 5–22Vβ subfamily T cells were found in samples from these patients whose transplantation more than one year. TCR repertoire complexity was abnormal in all patients. Discussion Following allogeneic BMT, regeneration of T-cell populations with a diverse repertoire can occure by at least two mechanisms: One mechanism is a thymic-dependent pathway, which presumably involves both negative and positive selection and recapitulates fetal ontogeny. Alternatively, regeneration of peripheral T cells may occur through thymic-independent mechanisms. All patients had marked abnormalities in their spectratypes, only 5-22Vβ subfamily T cells were found in samples from these patients, most of it was influenced after transplant, although the number of circulating CD3+ T lymphocytes in these patients have restored at normal lever by flow cytometic analysis, but the CD4+ T cell subset returned slowly in these patients resulting in an inversion of the normal CD4/CD8 ratio for more than 1 year after tuansplantation. Therefore, the analysis of TCRVβ subfamily is a usuaful methods and techniques for monitoring immune reconstitution after transplant.