Enhancement of anti-tumor immunity by tumor cells transfected with the secondary lymphoid tissue chemokine EBI-1-ligand chemokine and stromal cell-derived factor-1alpha chemokine genes

Several new lymphocyte-specific chemokines, which attract naive and memory T cells, B cells, dendritic cells and natural killer cells, have been isolated. We have found evidence of the anti-tumor effects of 3 major lymphocyte-specific chemokines, secondary lymphoid tissue chemokine (SLC), EBI-1-ligand chemokine (ELC) and stromal cell-derived factor (SDF)-1alpha, in murine models (Meth A fibrosarcoma and HM-1 ovarian tumor). In both naive and immunized mice, tumors expressing SLC, ELC or SDF-1alpha showed delayed progression compared with control tumors. In mice immunized with tumor cells expressing 1 of these 3 chemokine genes, challenge with parental tumor cells resulted in slightly slower progression than in control mice, while in mice immunized with tumor cells transfected to co-express IL-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as these chemokines, all tumors regressed. Furthermore, spleen cells from mice immunized with these "double-transfected" tumor cells exhibited higher proliferative responses and greater cytotoxic activity against parental tumor cells. These anti-tumor effects were associated with profound alterations in the leukocyte populations within the tumors and regional lymph nodes, and this was due to activation of type I T cell-dependent responses that produced high levels of IFN-gamma. These findings show that SLC, ELC and SDF-1alpha enhance anti-tumor immunity both systemically and locally and that these chemokines may be clinically useful, especially when combined with IL-2 and GM-CSF.     

Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene

Nucleic acid vaccines represent an attractive approach to generating antigen-specific immunity because of their stability and simplicity of delivery. However, there is still a need to increase the potency of DNA vaccines. Using human papillomavirus type 16 E7 as a model antigen, we evaluated the effect of linkage to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of antigen-specific immunity generated by naked DNA vaccines. We found that vaccines containing E7-HSP70 fusion genes increased the frequency of E7-specific CD8+ T cells by at least 30-fold relative to vaccines containing the wild-type E7 gene. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing tumors. Surprisingly, E7-HSP70 fusion vaccines exclusively targeted CD8+ T cells; immunological and antitumor effects were completely CD4-independent. These results indicate that fusion of HSP70 to an antigen gene may greatly enhance the potency of DNA vaccines via CD8-dependent pathways.     

Enhancement of DNA vaccine potency by coadministration of a tumor antigen gene and DNA encoding serine protease inhibitor-6

Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits granzyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells. We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1. In this study, we explored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8+ T-cell immune responses and E7-specific antitumor effects. This combination of strategies resulted in significantly increased E7-specific CD8+ T-cell and CD4+ Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6. Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8+ T cells ( approximately 5-fold). Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6. Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia.     

T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that is selective in its recruitment of naive T cells and dendritic cells (DCs). In the lymph node, SLC is believed to play an important role in the initiation of an immune response by colocalizing naive T cells with DC-presenting antigen. Here, we used SLC as a treatment for tumors established from the poorly immunogenic B16 melanoma. Intratumoral injections of SLC inhibited tumor growth in a CD8+, T cell-dependent manner. SLC elicited a substantial infiltration of DCs and T cells into the tumor, coincident with the antitumor response. We next used SLC gene-modified DCs as a treatment of established tumors. Intratumoral injections of SLC-expressing DCs resulted in tumor growth inhibition that was significantly better than either control DCs or SLC alone. Distal site immunization of tumor-bearing mice with SLC gene-modified DCs pulsed with tumor lysate elicited an antitumor response whereas control DCs did not. We also found that s.c. injection of lysate-pulsed DCs expressing SLC promoted the migration of T cells to the immunization site. This report demonstrates that SLC can both induce antitumor responses and enhance the antitumor immunity elicited by DCs.     

Enhancement of DNA vaccine potency by linkage of antigen gene to a gene encoding the extracellular domain of Fms-like tyrosine kinase 3-ligand

Recently, Flt3 (Fms-like tyrosine kinase 3)-ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells (APCs), particularly dendritic cells (DCs). A recombinant chimera of the extracellular domain of Flt3-ligand (FL) linked to a model antigen may potentially target the antigen to DCs and their precursor cells. Using human papillomavirus-16 E7 as a model antigen, we evaluated the effect of linkage to FL on the potency of antigen-specific immunity generated by naked DNA vaccines administered intradermally via gene gun. We found that vaccines containing chimeric FL-E7 fusion genes significantly increased the frequency of E7-specific CD8+ T cells relative to vaccines containing the wild-type E7 gene. In vitro studies indicated that cells transfected with FL-E7 DNA presented E7 antigen through the MHC class I pathway more efficiently than wild-type E7 DNA. Furthermore, bone marrow-derived DCs pulsed with cell lysates containing FL-E7 fusion protein presented E7 antigen through the MHC class I pathway more efficiently than DCs pulsed with cell lysates containing wild-type E7 protein. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing metastatic tumors. The FL-E7 fusion vaccine mainly targeted CD8+ T cells, and antitumor effects were completely CD4 independent. These results indicate that fusion of a gene encoding the extracellular domain of FL to an antigen gene may greatly enhance the potency of DNA vaccines via CD8-dependent pathways.     

Herpes simplex virus (HSV) amplicon-mediated codelivery of secondary lymphoid tissue chemokine and CD40L results in augmented antitumor activity

Development of effective antitumor immune responses depends on timely interactions of effector cells. A bimodal approach that involves coexpression of chemokines and costimulatory molecules within the tumor bed may elaborate a more optimal antitumor response. One candidate includes secondary lymphoid tissue chemokine (SLC), which promotes the colocalization of na?ve, nonpolarized memory T cells and dendritic cells (DCs) within lymph nodes and Peyer's patches. CD40L-mediated DC activation could induce maturation, enhance antigen presentation, and facilitate priming of the recruited na?ve T cells. To this end, the antitumor activity of SLC and CD40L expressed singly or in combination using the herpes simplex virus (HSV)-derived amplicon was examined in two murine models: A20, a B-cell lymphoma, and CT-26, an adenocarcinoma. Administration of amplicons encoding SLC (HSV-SLC) into s.c. tumors established previously resulted in heavy infiltration of CD4+ and CD8+ T cells, and DCs, and the generation of cytolytic T-cell activity. Combined transduction of either tumor with HSV-SLC and HSV-CD40L resulted in a more enhanced antitumor activity that was CD8+ T cell-dependent than observed with either vector alone. mRNA expression of the Th1 markers IFN-gamma, perforin, and interleukin 12 was detectable only in transduced regressing tumors. In addition to identifying a potent antitumor immune strategy, we show that amplicon-mediated SLC and CD40L delivery may mimic lymph node conditions necessary for priming na?ve T cells within the tumor bed, and demonstrate the importance of DC activation status on antigen presentation and cytokine expression for priming of newly recruited T cells.     

CCL21 (SLC) improves tumor protection by a DNA vaccine in a Her2/neu mouse tumor model

Secondary lymphoid-tissue chemokine (SLC/CCL21) is a CC chemokine that is constitutively expressed in various lymphoid tissues and binds to chemokine receptor CCR7 on mature dendritic cells (DCs) and distinct T-and B-cell sub-populations. In vivo, CCL21 regulates the encounters between DC and T cells and thus is a key regulator of adaptive immune responses. We asked whether CCL21 is able to augment immunogenicity of a DNA-based vaccine against Her2/neu in a Balb/c mouse model with syngeneic Her2/neu+ tumor cells (D2F2/E2). Mice were vaccinated intramuscularly with plasmid DNA (pDNA) on day 1 and boosted on day 15; tumor challenge was performed subcutaneously on day 25. Coexpression of CCL21 and Her-2/neu resulted in induction of a TH1-polarized immune response and substantial improvement of the protective effect of the DNA vaccine. Coexpression of tumor antigen pDNA(Her2/neu) with both pDNA(GM-CSF) and pDNA(CCL21) as adjuvants led to further improvement of protection by the vaccine (70% tumor-free mice on day 35 vs 40% with either adjuvant alone vs 5-10% with tumor antigen alone). Our results show that CCL21 is a potent adjuvant for DNA vaccination, particularly in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu/CCL21/GM-CSF) vaccine might be particularly promising in minimal residual Her2/neu+ breast cancer.     

c-myc gene mutation in gastric mucosa-associated lymphoid tissue (MALT) lymphoma and diffuse large B-cell lymphoma

The c-myc gene is involved in important cellular processes, including cell proliferation, differentiation, and apoptosis. We analyzed mutation of the c-myc gene in 51 patients with gastric lymphoma [27 patients with low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, 11 with high-grade MALT lymphoma, and 13 with diffuse large B-cell lymphoma (DLL)], by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. We also evaluated the relationship between mutation of the c-myc gene and regression of low-grade MALT lymphoma after Helicobacter pylori (H. pylori) eradication. Mutation in exon 2 of the c-myc gene was present in 2 of 20 (10%) patients with low-grade MALT lymphoma, in 1 of 7 (14%) patients with high-grade MALT lymphoma, and none of 10 patients with DLL. The 3 patients who had mutations of the gene, showed different patterns of mobility shift, suggesting different mutations. In addition, 15 patients with low-grade MALT lymphoma received anti-H. pylori therapy. All the patients achieved eradication. Nine of the 15 (60%) patients with low-grade MALT lymphoma showed complete regression (CR), 3 (20%) showed partial regression (PR), and 3 (20%) showed no change (NC). One of the 9 (11%) CR patients had a mutation of the c-myc gene. None of the 3 PR and 3 NC patients had mutation of the gene. There was no significant difference between the frequencies among the c-myc gene mutation in CR, in PR and in NC patients. These data suggest that mutation of the c-myc gene may not be commonly associated with development of gastric MALT lymphoma and DLL, and may not be associated with regression of low-grade MALT lymphoma after H. pylori eradication.     

Enhancement of DNA vaccine potency by antigen linkage to IFN‐γ‐inducible protein‐10

DNA vaccines have emerged as an attractive approach to generate antigen‐specific T‐cell immune response. Nevertheless, the potency of DNA vaccines still needs to be improved for cancer immunotherapy. In this study, we explored whether functional linkage of a Th1‐polarizing chemokine, IP‐10, to a model tumor antigen, human papillomavirus type 16 (HPV‐16) E7, enhanced DNA vaccine potency. IP‐10 linkage changed the location of E7 from the nucleus to the endoplasmic reticulum and led to the secretion of functionally chemoattractive chimeric IP‐10/E7 protein. In addition, this linkage drastically enhanced the endogenous processing of E7 antigen through MHC class I. More importantly, we found that C57BL/6 mice intradermally vaccinated with IP‐10/E7 DNA exhibited a dramatic increase in the number of E7‐specific CD4⁺ Th1 T‐cells and CD8⁺ T‐cells and, consequently, were strongly resistant over the long term to E7‐expressing tumors compared to mice vaccinated with wild‐type E7 DNA. Thus, because of the increase in tumor antigen‐specific T‐cell immune responses obtained through both enhanced antigen presentation and chemoattraction, vaccination with DNA encoding IP‐10 linked to a tumor antigen holds great promise for treating tumors.     

The clinical pathology and DNA ploidy of gastric mucosa associated lymphoid tissue (malt) lymphoma infiltrating the leiomyomas of the uterus

Themucosaassociatedlymphoidtissue(MALT)lymphomaisasortoflowermalignantlymphomawithspecialpathologicalmorphologyandclinicalcharacteristics.[l]Itsinvolvementinleimyomasoftheuterusisrelativelyrare.InthisarticlewereportonecaseofMALTlymphomainstomachanditscellsinfiltratedtheLeimyomas'intheuterusandandalsoreportitsclinicopathologycharacteristicsandDNAploidy.MATERIALSANDMETHODSThepatientwasa54yearsoldwoman.Shehadbeenmenopausaulfortwoyears.Then,shehadalossof10kilogramsofherbodyweightin4mo…     

胸腺粘膜相关淋巴瘤（病例报告及文献复习）

本文报道经免疫组化证实的8例B细胞性胸腺粘膜相关淋巴瘤,其特征为胸腺组织结构被中心细胞样小淋巴细胞浸润,但Hassall's小体仍可部分保存,并常形成含有分泌物的微囊,组成典型的淋巴上皮样病变,符合粘膜相关淋巴组织淋巴瘤的特征。免疫组化反应显示多数为MB2单抗反应阳性、λ或κ阳性的B细胞,属低度恶性淋巴瘤,对该瘤的组织发生,细胞成份,诊断和鉴别诊断进行了讨论,并指出应从胸腺瘤淋巴细胞型中单独分出来。     

Identification of differentially expressed genes in human gliomas by DNA microarray and tissue chip techniques

New genomic large-scale screening techniques have made the task of establishing an accurate molecular fingerprint of cancer cells feasible. Here, we have used a two-phase strategy for identification of molecular alterations in gliomas. First, cDNA microarrays (Clontech Laboratories, Inc., Research Genetics) were used to pinpoint differentially expressed genes between normal brain and diffuse astrocytomas (grades II-IV), and between a primary tumor and a later tumor reoccurrence in the same patient. More than 200 gene expression alterations were detected from glioblastomas, whereas relatively few changes were seen in grade II and grade III tumors. The most distinct progression-related expression change was the up-regulation of the insulin-like growth factor binding protein 2 (IGFBP2) gene. Second, a high-density tissue microarray of 418 brain tumors was constructed and used for clinical validation of gene expression changes. Strong expression of IGFBP2 was associated with progression and poor patient survival in diffuse astrocytomas (P < 0.0001). Third, comparisons of the data between (a) multiple spots retrieved from one predefined tumor region (IGFBP2 and vimentin immunohistochemistry, 20 tumors) or between (b) standard slides and arrayed tissues (p53 immunohistochemistry, 42 tumors) revealed very little variation. In conclusion, the combined use of DNA microarrays and tissue microarrays offers a powerful strategy for rapid identification and thorough characterization of differentially expressed genes in gliomas.     

Alkylation of rodent tissue DNA induced by N-nitrosobis(2-hydroxypropyl)amine.

Levels of methyl and hydroxypropyl adducts induced by single s.c. injections of various doses of tritium-labeled N-nitrosobis(2-hydroxypropyl)amine ([1-3H]BHP) were determined in the liver, pancreas, kidney and lung of hamsters and rats. At doses of BHP used in carcinogenesis studies (100-500 mg/kg), methylation of DNA was more extensive than its hydroxypropylation; however, it did not increase proportionally with the dose and gradually became secondary to hydroxypropylation at higher doses of the carcinogen. Ratios of hydroxypropyl versus methyl adducts also varied significantly depending on the tissue and species. In both species ratios of N7-hydroxypropylguanine (N7-HpG) versus N7-methylguanine (N7-MeG) were greater in kidney and pancreas than in liver or lung. Due to apparent differences in the repair of O6-methylguanine (O6-MeG) and O6-hydroxypropylguanine (O6-HpG), and the propensity of 2-hydroxypropylating as compared to methylating agents to yield a greater percentage of oxygen adducts, ratios of O6-HpG versus O6-MeG were markedly greater than those of N7-HpG versus N7-MeG. Levels of O6-HpG were greater than those of O6-MeG in rat liver, pancreas and kidney and also in hamster kidney, while such levels were similar in rat lung and also in hamster liver, pancreas and lung. Like N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP), BHP was activated primarily in the liver and induced substantially greater DNA damage in this than in any other tissue examined. However, unlike BOP and HPOP, which induced similar levels of hepatic DNA damage in the above two species, BHP methylated and hydroxypropylated hamster liver DNA more extensively than that of the rat. Differences between BOP and BHP were also observed regarding levels and distribution of DNA adducts in extrahepatic tissues. In rats, BHP induced greater levels of methylation and hydroxypropylation in lung than in kidney, while the reverse was observed with BOP. Apparently reduction of the beta-carbon of pancreas-specific nitrosamine carcinogens results in a shift of alkylation from kidney to the lung. Excretion of HPOP in the urine of BHP-treated animals and the observed saturation of DNA methylation at high doses of BHP, supported the hypothesis that the BHP-induced methylation of DNA proceeded via the intermediate formation of HPOP. This was further supported by the observation that both excretion of HPOP and levels of methyl adducts were greater in hamsters than in rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of apoptotic cells and immunohistochemical study of bcl-2 and p53 gene protein in primary gastric mucosa-associated lymphoid tissue (MALT) lymphoma

In recent years, the study of apoptosis has become one of the hot subjects for cancer research. Recent research revealed that apoptosis might be associated with tumor initiation and development, cancer therapy and prognosis. A lot of genes have been found to involve in the apoptotic process, especially the p53 and bcl-2 gene. The mucosa associated lymphoid tissue type lymphoma (MALToma) is a sort of extra-nodal low-grade malignant lymphoma, which is different from primary nodal lymphomas in …     

Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes

Repair of alkylated bases in DNA is performed by O⁶-methylguanine-DNAmethyltransferase (MGMT) and a set of enzymes of the base excisionrepair pathway involving N-methylpurine-DNA glycosylase (MPG),apurinic endonuclease (APE), DNA polymerase ß (Polß) and DNA ligase. The level of expression of theseenzymes may exert a profound effect on resistance of cells towardsalkylating drugs. We have comparatively analyzed the expressionof MGMT and the different base excision repair genes in rathepatoma cells (line H4IIE) after exposure to alkylating agents,X-rays and the glucocorticoid hormone dexamethasone. Furthermore,the effect of these agents on the activity of the cloned humanMGMT promoter was assayed. Exposure of cells to N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) or ionizing radiation increased MGMT mRNA levels up to4.5-fold. Under the same conditions of treatment, exerting onlya weak toxic effect, MPG and DNA ligase I mRNA levels were notenhanced, whereas the amounts of APE and Pol ß mRNAtransiently increased by     

A combination of flk1-based DNA vaccine and an immunomodulatory gene (IL-12) in the treatment of murine cancer

AIM: The aim of this study was to investigate the antivasculature effects and the antitumor effects of combining attenuated Salmonella typhimurium vaccine strain encoding murine vascular endothelial growth factor (VEGF) receptor-2 (flk1) with plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. METHODS: Mouse models of Gl261 glioblastoma were treated with combining orally given attenuated Salmonella typhimurium vaccine strain encoding flk1 with direct intratumoral injection of a nonviral plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. The volumes of tumors were observed. Cytolytic T lymphocyte (CTL) response was measured by a 4-hour 51Cr release assay, vessle density and tumor cell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNEL staining. RESULTS: Compared to mice receiving single agent therapy, received either oral immunization flk1-based vaccine only or the therapeutic gene-IL-12 plasmid DNA only or those in the control group, the combination therapy groups developed a strong CTL response and showed more significantly inhibited tumor growth, apoptosis of tumor cells, and reduced neovascularization and cell proliferation in these mice. CONCLUSIONS: The therapy of attenuated Salmonella typhimurium vaccine strain encoding flk1 combined with the interleukin-12 gene has significant synergistic effect against tumors.     

Nongastrointestinal mucosa-associated lymphoid tissue (MALT) lymphomas: Clinical and therapeutic features of 24 localized patients

Background: Peripheral B-cell lymphoma of the marginal zone (MALT, low-grade), presenting as localized, extranodal disease, usually affects the elderly. The gastrointestinal tract is the most frequently involved extranodal location, representing 70% of all MALT lymphomas. Recently, numerous other extranodal sites involved by MALT lymphomas have also been described.Patients and methods: From January 1990 to October 1995, 24 patients with untreated nongastrointestinal low-grade MALT lymphoma were submitted to treatments ranging from the local approach of radiotherapy and local -interferon (-IFN) administration to chemotherapy. The tumours were located in the lung (seven cases), conjunctiva (four cases), lachrymal gland and orbital soft tissue (four cases), salivary glands (three cases), skin (three cases), breast (two cases)' and thyroid (one case). All patients had low-grade stage IE tumours.Results: Chemotherapy was administered in 11 patients (six with lung, three with salivary gland, one with breast, and one with thyroid locations); radiation therapy was employed in seven patients (three with lachrymal gland, three with skin, and one with breast locations); local -IFN administration was administered in five patients (four with conjunctival, and one with lachrymal gland sites); and surgery was employed in one patient with a lung tumour. All patients achieved complete remissions; three local recurrences and two relapses in other sites were observed. The global five-year survival rate was 100% with a relapse-free survival rate of 79%.Conclusions: These data confirm the significant efficacy of different therapeutic approaches to specific sites inbes obtaining a good remission rate for nongastrointestinal localized low-grade MALT lymphomas.     

THE IMMUNOHISTOCHEMISTRY AND QUANTITATIVE MORPHOMETRIC STUDY OF MUCOSA ASSOCIATED LYMPHOID TISSUE (MALT) LYMPHOMA IN STOMACH

Objective: To invetigate the Immunohistochemistry characters and quantitative nmorphometric significance for the mucosa associated lymphoid tissue (MALT) lymphoma of stomach in 14 patients. Methods: The routine paraffin slides were cut, stained with H.E., and immunochemically by ABC method. The morphologic appearance of nuclei of lymphoma cells were measured with HPIAS-1000 color pathology picture analysis system. Results of the 14 cases, 9 was centrocyte like (CCL) cell lymphoma, 2 CCL with large cell lymphoma, 1 small no cleaved cell lymphoma, 1 large no cleaved cell lymphoma, 1 T immunoblastic malignant lymphoma. The morphologic measurement results showed that there were great significant differences (P<0.001) for the 15 items of morphology parameters between the nuclei of MALT lymphoma cells and those of normal control lymphocytes in stomach. There were great significance differences (P<0.001) or significance (P<0.05) for the most of the 15 items of morphologic parameters of nuclei among the 5 types of MALT lymphoma. Especially, that the values of area, circumference, equivalent diameter, area volume, circumference volume, long diameter, short diameter, practical area were increasing as the malignant degree of classification was rising, which reflect the increasing malignancy of the tumor. Conclusion: It was suggested that with the quantitative morphology measurement method, man could make accurate diagnosis for MALT lymphoma. It offered us a new method to make the diagnosis, so that it had significance. It might be also practicable with morphology measurement method to make the sub classification of MALT lymphoma.