Splenic marginal zone lymphoma-like features in API2-MALT1 transgenic mice that are exposed to antigenic stimulation

Recently, we described a transgenic mouse model to analyze the effect of the API2-MALT1 fusion-protein in vivo. Our results showed that the expression of API2-MALT1 is not sufficient to induce the development of lymphoma masses. Here, we demonstrate that immunization with Freund's complete adjuvant led to the loss of compartmentalization of the splenic white pulp in API2-MALT1 transgenic mice, resulting in a splenic marginal zone lymphoma-like lymphoid hyperplasia of a peculiar B-cell subset that disappeared as soon as the antigenic stimulation faded away. These data indicate an effect of API2-MALT1 expression on the normal immune response.     

Monitoring gastric lymphoma in peripheral blood by quantitative IgH allele-specific oligonucleotide real-time PCR and API2-MALT1 PCR

Gastric extranodal marginal zone lymphoma (EMZL) often shows prolonged localised disease, but the present study demonstrated the presence of tumour cells in peripheral blood (PB) of low stage patients. We studied the presence of tumour cells in PB in gastric lymphoma patients harbouring or lacking t(11;18)(q21;q21), by real-time immunoglobulin (Ig)H allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) and API2-MALT1 PCR. Tumour cells were exclusively detected in PB of t(11;18)(q21;q21)+-EMZL patients. The presence of tumour cells in PB and gastric biopsy follow-up samples showed a good correlation in these patients, suggesting clinical relevance for monitoring of tumour cells in PB of gastric t(11;18)(q21;q21)+-EMZL patients.     

A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression.

The locus control region of the human beta-globin cluster consists of four major DNase I hypersensitive sites (HS). When linked to globin genes, the locus control region confers a high level of erythroid-specific expression of these genes in transgenic mice or transfected erythroid cell lines. We have examined the effect of one of these sites, HS2, on human beta-globin gene expression in a murine erythroleukemia cell line (MEL) after retrovirus-mediated gene transfer. We incorporated a 732- or 412-base-pair (bp) segment of HS2 in the retroviral construct carrying the human beta-globin gene. These fragments rendered the viruses unstable as the human beta-globin gene was rearranged or deleted in all the packaging cell lines examined. On the other hand, when a 36-bp fragment containing the NFE-2/AP-1 binding consensus in this region was inserted into the retroviral construct, we recovered 6 stable packaging cell lines of 12 examined, similar in percentage to the construct with the beta-globin gene alone. The virus titers of the packaging cell lines from these two constructs were similar. We infected MEL cells with viruses produced from three packaging cell lines of each of the two constructs and measured the ratio of human beta-globin to mouse alpha-globin mRNA after hexamethylenebisacetamide induction. The overall level of expression increased 2-fold from 6.0% to 12.7% with the addition of this 36-bp enhancer.     

H. pylori negative gastric MALT lymphoma with API2-MALT1 translocation treated by endoscopic submucosal dissection: A case report

Rationale:API2-MALT1 positive gastric mucosa-associated lymphoid tissue (MALT) lymphomas are considered to have favorable prognosis. We report a case of API2-MALT1 positive gastric MALT lymphoma, treated by endoscopic submucosal dissection (ESD).Patient concerns:A 51-year-old man underwent esophagogastroduodenoscopy (EGD) for the annual health checkup examination.Diagnoses:The EGD showed a reddish depressed lesion with small reddish spots in the lower gastric body. There was no endoscopic atrophy in the entire stomach and Helicobacter pylori (H. pylori) serum test was negative. Infiltration of small lymphocytes was shown in the gastric tissues obtained by the endoscopic biopsy. The fluorescence in situ hybridization using the biopsy samples confirmed the presence of genetic translocation of API2-MALT1, suggesting that the lesion is API2-MALT1 positive MALT lymphoma.Interventions:Since endoscopic ultrasound suggested that the lesion was localized within the lamina propria mucosae, we performed ESD to achieve the en bloc resection of the lesion.Outcomes:Conclusive diagnosis of gastric MALT lymphoma was made based on the resected specimen. Lateral and vertical margins were negative. No lymphoma cells were detected using endoscopic biopsy after 5 years.Lessons:Our report suggests that ESD can be considered as alternative treatment for API2-MALT1 positive gastric MALT lymphoma if the lesion was localized within the gastric mucosa.     

Clinical features and prognosis of gastric MALT lymphoma with special reference to responsiveness to H. pylori eradication and API2-MALT1 status

BACKGROUND AND AIM: Clinicopathologic characteristics and prognosis of Helicobacter pylori eradication-resistant gastric MALT lymphoma have not been well clarified. We analyzed a consecutive series of gastric MALT lymphomas at our institution regarding treatment, clinical course, and prognosis, with special reference to responsiveness to H. pylori eradication and presence of API2-MALT1. METHODS: Subjects were 92 consecutive patients with gastric MALT lymphoma. Seventy were H. pylori positive, and 87 received H. pylori eradication therapy. The remaining five cases were API2-MALT1 positive and did not receive eradication treatment. Second-line treatments were radiation therapy, total gastrectomy, and chemotherapy (rituximab, rituximab plus CHOP, or rituximab plus 2-chlorodeoxyadenosine). RESULTS: Gastric MALT lymphoma was classified into three groups, except one case with API2-MALT1 who responded to H. pylori eradication therapy: responders without API2-MALT1 (group A, N = 56, 65%), nonresponders without API2-MALT1 (group B, N = 16, 19%), and nonresponders with API2-MALT1 (group C, N = 14, 16%). Most cases in group A attained complete remission (CR) in 2 or 3 months and CR persisted for an average of 51.1 months (3-134 months). Recurrence was only seen in one case. In groups B and C, radiation therapy, chemotherapy, and total gastrectomy resulted in CR in 13, 5, and 2 cases, respectively. In 5 group B patients and 6 group C patients who did not undergo second-line therapy, disease did not progress for an average of 10.4 and 40.1 months, respectively. In 1 group C case who did not receive second-line treatment, lymphoma metastasized to the lung 12 yr after eradication. All group B patients and all but 2 group C patients remain alive; one of these deaths was from gastric carcinoma developing 7 yr after eradication. CONCLUSION: Gastric MALT lymphoma responding to H. pylori eradication demonstrated good prognosis, and for nonresponsive cases, second-line treatments resulted in CR. However, careful observation for development of gastric carcinoma and disease progression is essential during follow-up of API2-MALT1-positive MALT lymphoma when patients decline second-line treatment.     

A case of H. pylori and API2-MALT1 gene-negative gastric mucosa-associated lymphoid tissue lymphoma with a carcinoma-like signet-ring cell lymphoepithelial lesion]

A 60-year-old woman presented in February 2003 with an ulcer on the lesser curvature of the anglus. The endoscopic biopsy specimens showed epithelial signet-ring cell associated with lymphoid infiltration, suggesting a diagnosis of gastric cancer. Histopathological examination confirmed the diagnosis of low-grade B-cell lymphoma of MALT with epithelial signet-ring cell lymphoepithelial lesion, which was negative for H. pylori and t (11;18) (q21;q21) translocation (API2-MALT1 gene). This case was treated with H. pylori eradication and additional radiation therapy, and the tumor was disappeared.     

肺黏膜相关B淋巴细胞淋巴瘤中BCL10蛋白表达和染色体易位的关系

目的 检测肺黏膜相关B淋巴细胞淋巴瘤(MALT淋巴瘤)中BCL10蛋白表达及目前报道的染色体易位的发生率.包括t(11;18)/AP12-MALTI、t(1;14)/BCLIO-lgH及t(14;18)/MALTI-IgH.探讨BCL10异常表达和染色体易位的关联程度.方法 收集复旦大学附属肿瘤医院病理科2000～2007年诊断的23例肺MALT淋巴瘤患者的病理组织蜡块,其中男19例,女4例,年龄27～84岁,平均55.8岁.用免疫组织化学EnVision法检测BCL10蛋白的表达,用荧光原位杂交(FISH)法分别检测API2-MALTI、BCL10、MALTl和IgH基因的异常,并对可联络到的10例病例进行随访.结果 23例中19例BCL10蛋白表达阳性,其中细胞质阳性9例,细胞核阳性者10例;用FISH方法榆测了全部病例,其中9例可检测到API2-MALTI融合基因.1例町能为BCL10-IgH融合,未发现IgH-MALT1基因异常.细胞核BCL10蛋白表达阳性的10例中,仅5例同时伴有基因异常;BCL10异常核表达与染色体易位无明显相关性(x2=0.306,P=0.685).有随访资料的10例患者全部生存(随访时间7～35个月),但治疗方式各异(单纯化疗、手术或手术加化疗).结论 肺MALT淋巴瘤患者组织内BCL10在细胞核中表达率较高,t(11;18)/AP12-MALT1融合基因是肺MALT淋巴瘤中最常见的染色体异常,而t(1;14)/BCL10-IgH和t(14;18)/MALT1-IgH在肺MALT淋巴瘤中不常见;核表达BCL10与发生染色体异常是瓦相独立的凶素,但这些特点对肺MALT淋巴瘤的诊断有一定的辅助价值,特别是对纤维支气管镜活检小标本组织的诊断更有帮助.     

MALT1 and the API2-MALT1 fusion act between CD40 and IKK and confer NF-kappa B-dependent proliferative advantage and resistance against FAS-induced cell death in B cells

The most frequently recurring translocations in mucosa-associated lymphoid tissue (MALT) B-cell non-Hodgkin lymphoma, t(11;18)(q21;q21) and t(14;18)(q32; q21), lead to formation of an API2-MALT1 fusion or IgH-mediated MALT1 overexpression. Various approaches have implicated these proteins in nuclear factor kappaB (NF-kappa B) signaling, but this has not been shown experimentally in human B cells. Immunohistochemistry showed that MALT1 is predominantly expressed in normal and malignant germinal center B cells, corresponding to the differentiation stage of MALT lymphoma. We expressed MALT1 and apoptosis inhibitor-2 API2/MALT1 in human B-cell lymphoma BJAB cells and found both transgenes in membrane lipid rafts along with endogenous MALT1 and 2 binding partners involved in NF-kappa B signaling, B-cell lymphoma 10 (BCL10) and CARMA1 (caspase recruitment domain [CARD]-containing membrane-associated guanylate kinase [MAGUK] 1). API2-MALT1 and exogenous MALT1 increased constitutive NF-kappa B activity and enhanced I kappa B kinase (IKK) activation induced by CD40 stimulation. Both transgenes protected BJAB cells from FAS (CD95)-induced death, consistent with increases in NF-kappa B cytoprotective target gene expression, and increased their proliferation rate. Expression of a dominant-negative I kappa B alpha mutant showed that these survival and proliferative advantages are dependent on elevated constitutive NF-kappa B activity. Our findings support a model in which NF-kappa B signaling, once activated in a CD40-dependent immune response, is maintained and enhanced through deregulation of MALT1 or formation of an API2-MALT1 fusion.     

Antiapoptotic effect of endothelin-1 in rat cardiomyocytes in vitro

Apoptosis of cardiac myocytes is thought to be a feature of many pathological disorders, including congestive heart failure (CHF) and ischemic heart disease (IHD). Because recent investigations indicate that endothelin-1 (ET-1) plays an important role in CHF and IHD, we investigated the effect of ET-1 on cardiomyocyte apoptosis. The presence of apoptosis in rat cardiomyocytes (H9c2 and neonatal) was evaluated by morphological criteria, electrophoresis of DNA fragments, 4',6'-diamidine-2'-phenylindole staining, and TUNEL analysis. ET-1, but not angiotensin II, prevented apoptosis induced by serum deprivation via ETA receptors in a dose-dependent manner (1 to 100 nmol/L). ET-1 also prevented cytochrome c release from mitochondria to the cytosol. The use of specific pharmacological inhibitors demonstrated that the antiapoptotic effect of ET-1 was mediated through a tyrosine kinase pathway (genistein and AG490) but not through protein kinase C (PKC; calphostin C), mitogen-activated protein kinases (PD98059 and SB203580), or PKA (KT5270) pathways. Adenovirus-mediated gene transfer of kinase-inactive (KI) c-Src reversed the antiapoptotic effect of ET-1. We further investigated whether Bcl-xL, an antiapoptotic molecule, would be upregulated by using a luciferase-based reporter system. ET-1 upregulated Bcl-xL, and this upregulation was inhibited by genistein or AG490 but not by calphostin C. The experiments with KI mutants for various tyrosine kinases revealed that c-Src and Pyk2 (but not JAK1, Jak2, Syk, and Tec) are involved in ET-1-induced upregulation of Bcl-xL expression. These findings suggest that ET-1 prevents apoptosis in cardiac myocytes through the ETA receptor and the subsequent c-Src/Bcl-xL-dependent pathway.     

Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer

We have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells. The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent- activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony- forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. Our results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and should provide a unique tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells.     

T淋巴细胞瘤转基因瘤苗抗肿瘤作用研究

目的观察C57小鼠源性淋巴瘤瘤苗抗肿瘤免疫效果。方法采用电穿孔法将粒细胞—巨噬细胞集落刺激因子（GM-CSF）表达质粒导入T淋巴瘤RMA细胞制作转基因瘤苗,筛选出高表达GM-CSF的细胞克隆（RMA-GM）,经丝裂霉素处理后皮下接种C57小鼠,观察抗肿瘤免疫效果。结果RMA-GM接种C57小鼠后,出瘤时间延长,肿瘤生长速度减慢,生存期延长,40%的小鼠长期生存。结论GM-CSF基因质粒转导的RMA-GM转基因瘤苗具有较好的抗肿瘤免疫效果。     

Expression of the nlsLacz gene in dendritic cells derived from retrovirally transduced peripheral blood CD34+ cells

BACKGROUND AND OBJECTIVE: Gene transfer and expression of exogenous genetic information coding for an immunogenic protein in antigen presenting cells (APCs) can promote an immune response. This was investigated by retroviral transfer of a marker gene into CD34+ derived APCs. DESIGN AND METHODS: To achieve long term expression of a specific transgene in APCs, G-CSF mobilized peripheral blood CD34+ cell populations were retrovirally transduced with the bacterial nlsLacZ, a marker gene used here as a model, in the presence of IL-3, IL-6, GM-CSF and SCF prior to being induced to differentiate into dendritic and macrophage cells by GM-CSF and TNF-a. RESULTS: Addition of IL-4 was found to induce dendritic differentiation preferentially by inhibiting proliferation and differentiation of the macrophage lineage. As assessed by X-Gal staining, LacZ gene expression was observed in cells from both the dendritic lineage (CD1a+/CD14-) which still exhibits the highest immunostimulatory activity in mixed lymphocyte reaction and from the macrophage lineage (CD1a-/ CD14+). INTERPRETATION AND CONCLUSIONS: This study sets out the possibility of transducing dendritic and macrophage progenitors present in the CD34+ cell population and in using a marker gene such as nlsLacZ to study gene expression in antigen presenting cell compartments.