Cytotoxicity Receptors Research Articles

Prospective study of chemotherapy-induced clostridium difficile infection in lung cancer patients.

e19521 Background: Diarrhea after antibiotic treatment is primarily ascribed to the development of mucosal lesion for which Clostridium difficile infection (CDI) is held responsible. Evidence on CDI in patients on chemotherapy without antibiotic treatment prior to chemotherapy is on the rise. However there have been few reports describing diarrhea in relation to regimens of chemotherapy. The aim of our study was to find out whether the incidence of CDI in patients on cancer chemotherapy increases even in the absence of prior antibiotic treatment. Methods: From March 2010 through September 2011, we investigated the presence of Clostridium difficile (C.difficile) and its toxin in any patients undergoing diarrhea while receiving cytotoxic anticancer agents or epidermal growth factor receptor tyrosine kinase inhibitors. Whenever Grade2 or worse diarrhea defined by the Common Terminology Criteria for Adverse Events version4 occurred, we examined their stool samples for anaerobic culture and simultaneously tested for toxins A and B. A case of CDI is defined by the presence of symptoms (usually diarrhea) and either a stool test positive for C.difficile toxins or toxigenic C.difficile according to criteria of the Society for Healthcare Epidemiology of America and the Infectious Diseases Society of America. Results: A total of 345 consecutive patients (492 in terms of chemotherapy -regimens) were enrolled in this study. 36 of 492 regimens (7.3%) showed Grade2 or worse diarrhea. CDIs without prior antibiotic exposure were confirmed in as many as 8 out of these 36 regimens (22.2%). CDI was more frequently observed in patients of the regimens including S-1 or irinotecan than other anticancer drugs. Conclusions: CDI may remain undetected among patients who had diarrhea defined as above unless due attention is paid to this possibility. It is highly recommended to perform tests for the detection of C.difficile toxins so that necessary measures can be instituted without delay in the treatment of diarrhea of patients on chemotherapy. Further analysis for drugs specificity and prevention against CDI is necessary.

NKG2 Receptors Acquired by Activated Human CD4 T Cells Are Involved in Oligodendrocyte Destruction in the Context of MS (P07.086)

Objective: To characterize novel mechanisms by which CD4 T cells can injure human oligodendrocytes, and identify these cells in patients with Multiple Sclerosis (MS). Background Destruction of oligodendrocytes is a fundamental hallmark of MS. Although CD4 T cells are considered important contributors to this immunopathology, the mechanisms involved are not completely resolved. In recent years, CD4 T cells that acquire NK-associated markers have been implicated in tissue destruction in other immunopathologies such as Crohns Disease. We investigated whether cytotoxic NKG2+ CD4 T cells were detected in MS patients and whether human adult oligodendrocytes express the recognized ligands. We studied whether these receptors were involved in the immune-mediated injury of oligodendrocytes. Design/Methods: Proportions of NKG2D/C+ CD4 T cells in the blood of MS patients versus healthy donors were analyzed using flow cytometry (FACS). Peripheral blood mononuclear cells (PBMCs) from human donors are activated with the lectin phytohemagglutinin (PHA) and stained for NKG2C, NKG2D, CD4. CD4 T cells isolated from these cultures are incubated with primary cultures of human adult oligodendrocytes, and then analyzed by FACS for effector functions. Immunohistochemistry was done on post-mortem CNS sections of MS patients. Results: Our studies show an increased proportion of cytotoxic NKG2D/C+ CD4 T cells in the blood of MS patients. These CD4 T cells expressed elevated levels of lytic enzymes. We have previously shown that inflamed human oligodendrocytes upregulate NKG2D ligands; we now show that they can also express NKG2C ligand (HLA-E) upon inflammatory conditions mimicking MS lesions. Our functional results show that blocking NKG2C and NKG2D on PHA-activated CD4 T cells decreases their cytotoxicity towards the oligodendrocytes. Our in situ studies show that these cells are detected in post-mortem CNS sections of MS patients. Conclusions: Our results demonstrate that activated human CD4 T cells can mediate injury to oligodendrocytes through acquired cytotoxic NK-associated receptors. Supported by: Multiple Sclerosis Society of Canada (MSSOC). Disclosure: Dr. Zaguia has nothing to disclose. Dr. Antel has received personal compensation for activities with Sanofi-Aventis Pharmaceuticals, Inc., Biogen Idec, Novartis, Serono, Inc., Genzyme Corporation, Teva Neuroscience, and GlaxoSmithKline. Dr. Antel has received personal compensation in an editorial capacity for Multiple Sclerosis. Dr. Antel has received research support from Novartis. Dr. Saikali has nothing to disclose. Dr. Arbour has nothing to disclose.

Abstract 5461: Role of the NLRP3 inflammasome in the development and drug resistance of malignant mesothelioma

Abstract Inflammation plays an important role in development of various cancers including malignant mesothelioma (MM). We have shown that asbestos activates NOD-like receptor protein 3 (NLRP3), a component of the inflammasome in human macrophages. As chronic asbestos exposure is a key risk factor for the development of MM, we hypothesized that inflammasome-mediated inflammation might underlie the pathogenesis of this cancer. To show the involvement of NLRP3 in asbestos-induced mesothelioma, we demonstrated that exposure of asbestos to immortalized human mesothelial cells (LP9/hTERT), a cell type responsible for origin of MM, caused mRNA increase and activation of NLRP3 as measured by caspase-1 activation and IL-1β release. Inhibition of NLRP3 by siRNA caused significant decreases in NLRP3 mRNA levels as well as asbestos-induced IL-1β release in medium. On the other hand, human MM lines and tumor tissues showed significantly decreased levels of NLRP3 and caspase-1 as compared to LP9 or matching normal tissues respectively. Our findings suggest that initial exposure to asbestos causes increased mRNA levels and activity of NLRP3, which may help in MM development by promoting mesothelial cell transformation. However, tumor development culminates in MM with decreased NLRP3 protein and increased drug resistance which may be due to caspase-1 inactivation. Thus NLRP3 may be an appropriate target for therapy of MM, especially in combination with cytotoxic chemotherapeutic drugs and IL-1 receptor antagonists. This study is supported by a Meothelioma Applied Research Foundation (MARF) grant (AS) and by NIEHS grants T32 ES07122 (BM). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5461. doi:1538-7445.AM2012-5461

Activation of NK cell granulysin by mycobacteria and IL-15 is differentially affected by HIV

NK cells play an important role in innate immunity to mycobacteria and are a significant source of the bactericidal effector molecule granulysin. Defects in NK cells have been described in HIV-infected patients, though mechanistic studies have focused on effector molecules relevant to anti-viral, and not anti-bacterial, function. Here we used primary NK cells from healthy human donors and an in vitro system to identify the phenotype of granulysin expressing NK cells, characterize activation stimuli that regulate granulysin, and to study the immediate effects of HIV on innate activation of NK cell granulysin expression. We observe that granulysin expression is co-associated with cytotoxicity receptors (NKp46, NKG2D) known to have important function in the cytotoxic response to M.tb-infected macrophages. Granulysin expression is significantly increased following exposure to IL-15 or Mycobacterium bovis BCG, but in contrast to our previous findings with CD8(+)T cells, expression is weakly activated by IL-21. Infection of PBMC with HIV-1 suppresses NK cell induction of granulysin by IL-15, but does not impair activation by BCG. These effects of HIV-1 are associated with reduced STAT5 phosphorylation in the IL-15 activated signaling cascade. These observations suggest that HIV may impair the anti-bacterial function of NK cells and have implications for clinical use of IL-15 to augment innate cell mediated immunity in HIV+ patients.

ERCC1 and RRM1 but not EGFR gene expression is predictive of shorter survival in advanced non-small cell lung cancer (NSCLC)

10030 Background: Pivotal studies indicate a role of excision repair cross-complementation 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) in conferring a differential sensitivity to cytotoxic chemotherapy and epidermal growth factor receptor (EGFR) has been recently deeply investigated in NSCLC. Methods: We retrospectively collected 70 formalin-fixed paraffin-embedded (FFPE) bronchoscopic/fine needle aspiration biopsies of NSCLC to investigate the expression levels of ERCC1, RRM1 and EGFR by Real-Time PCR (Lord R et al. Clin Cancer Research 2002, 8:2286–91). Results were correlated with survival using the Kaplan-Meier method. Results: Sixty-one (87%) specimens were successfully amplified. Median age was 62 years (range 26–75), male/ female ratio 44/17, stage III/IV 20/41; 43 patients received cisplatin-based chemotherapy; overall median survival (MS) was 13.3 months over a median follow-up time of 45 months. ERCC1 expression level ranged from 0.70 to 15.12, RRM1 0.60–17.82. By adopting cut-off values according to median expression levels, we found a strong correlation between ERCC1 and RRM1 mRNA levels (r=0.410; p<0.001). MS in patients with low ERCC1 was significantly longer (16.9 vs 11.3 months, p<0.006) as well as in patients with low RRM1 (13.9 vs 10.9 months, p<0.03). Concomitant high expression levels of ERCC1 and RRM1 (n=26) are predictive of a worse outcome (13.9 vs 10.9 months, p<0.05). Among patients treated with cisplatin-based regimens, low ERCC1 levels were also predictive of a significantly longer MS (23.0 vs 11.6 months, p<0.002). A lower median ERCC1 level (3.2 vs 4.7) and a correlation with a better outcome were also observed in females vs males. No correlation between gene expression levels and histology was reported. No significant correlation between EGFR expression levels (range 0.5–85.8) and survival was found, even when different cut-off values were tested. Conclusions: This retrospective study further validates ERCC1 and RRM1 as good candidates genes to customize chemotherapy. Prospective studies based on the selection of patients according to genes expression levels are a research priority in early and advanced stages of NSCLC. [Table: see text]

Mechanisms of adenosine‐induced cytotoxicity and their clinical and physiological implications

Extracellular ATP (ATPo) and adenosine are cytotoxic to several cancer cell lines, suggesting their potential use for anticancer therapy. Adenosine causes cytotoxicity, either when added exogenously or when generated from ATPo hydrolysis, via mechanisms which are not mutually exclusive and which involve, adenosine receptor activation, pyrimidine starvation and/or increases in intracellular S-adenosylhomocysteine: S-adenosylmethionine ratio. Given that adenosine also appears to protect against cytotoxicity via mechanisms including immunity against damage by oxygen free radicals, an understanding of the contribution of adenosine to ATPo-induced cytotoxicity is thus crucial, when considering any potential therapeutic use for these compounds. However, such an understanding has been largely hindered by the fact that many studies have not focused enough on the possibility that both ATPo and adenosine may mediate cytotoxicity in the same system. Such studies can benefit from use a range of ATPo concentrations when assessing the contribution of adenosine to ATPo-induced cytotoxicity. Whilst future molecular and pharmacological studies are needed to establish the nature of the cytotoxic adenosine receptor, it is possible that more than just one adenosine receptor type is involved and that the cytotoxic receptor(s) type is more likely to have a low affinity for adenosine. Activation of the adenosine receptor(s) would thus lead to cytotoxicity only at relatively high adenosine concentrations, while lower adenosine concentrations mediate non-cytotoxic physiological effects.

450. Anti-CD22 Immunolipoplexes Enhance Transduction of Adenovirus in B Lymphoma Cells Independent on Adenovirus' Receptor but Do Not Enhance the Cytotoxicity of Oncolytic Adenovirus

Adenovirus vectors have several features that make them attractive for potential use in gene therapy, and attention has focused particularly on replication-competent adenoviruses for cancer applications. These have been evaluated extensively in epithelial tumours but only recently in lymphoid malignancies, mainly due to the known resistance of the lymphoid lineage to adenovirus infection. However, we and other groups have shown that this resistance is not absolute and that adenovirus can infect primary chronic lymphocytic leukaemia (CLL) and B-cell lymphoma lines, and that infection with replication-competent human adenovirus results in cytotoxicity that varies among different patients and different lymphoma cell lines. We found that the cytotoxicity of Adenovirus to Burkitt's lymphoma cells correlates with the infectability of these cells to adenovirus, which depends on the expression of adenovirus receptors such as CAR and heparan sulfate glycosaminoglycans. In a panel of lymphoma cell lines including Jiyoye, Raji, Daudi/ICRF, Daudi/100k, P3HR1, BL-74 and CA-46, only Jiyoye and Raji showed a significant cytotoxicity and Adenovirus receptor expression. Most of these cell lines show a very low or no cytotoxicity to replication-competent adenovirus due to low expression of adenovirus receptors. Most lymphoma cells exhibit high expression of CD22, a membrane protein that is endocytosed when anti-CD22 antibody binds the protein. Based on these observations, an adenovirus-retargeting system was developed for CD22-mediated cell entry: adenovirus-containing immunolipoplexes formed with monoclonal anti-CD22 antibody were tested for enhanced transduction of adenovirus receptor-negative lymphoma cell lines (BL-74 and CA-46). The results demonstrate that the method increased adenovirus transduction by 95.4% (anti-CD22-immunolipoplexes vs naked adenovirus) and 62.3% (anti-CD22-immunolipoplexes vs lipid-adenovirus complex) in CA-46 cells, and 38.2% (anti-CD22-immunolipoplexes vs naked adenovirus) and 27.1% (anti-CD22-immunolipoplexes vs lipid-adenovirus complex) in BL-74 cells, respectively. However, the method did not increase the cytotoxicity of replication-competent adenovirus to these cells. In conclusion, this study demonstrates that anti-CD22 immunolipoplexes represent an efficient strategy to enhance adenovirus transduction independent of adenovirus receptor status. This protocol may have an impact on cancer therapy for lymphomas.

"Self" to cytotoxic T cells has to be 1,000 or less high affinity nonapeptides per MHC antigen.

In order to serve as the effective target of a relevant cytotoxic T-cell receptor, the same peptide fragment has to occupy at least 0.1% of the class I major histocompatibility complex (MHC) antigen sites on the plasma membrane. Because of this need, I content that the thymic educator cell of "self" to cytotoxic T cells can suppress autoreactive T-cell clones only with regard to at the most, 1,000 self nonapeptides per a given allelic form of class I MHC antigens; e.g., HLA-A2. Each allelic form of class I MHC antigen apparently developed the preferential binding affinity toward a specific set of nonapeptides. The requirement for preferential binding can either be permissive or stringent. In the case of human HLA-A2, those nonapeptides having either Leu or Met at the second position and mainly Val, but occasionally Leu at the ninth position are preferred. Since both Leu and Val are very common residues, the typical somatic cell type readily supplies nearly 3000 high affinity host nonapeptides preferred by HLA-A2. Of those, the tolerance can be induced, at the most, to only 1,000 nonapeptides. In view of this, permissive class I MHC antigens such as HLA-A2 carefully avoid high affinity nonapeptides in viral proteins, for their status as to self or nonself is uncertain, and they choose second choice nonapeptides as T epitopes. In sharp contrast to human HLA-A2, mouse H-2Db represents the stringent class I MHC antigens. In order to show the high binding affinity toward H-2Db, nonapeptides are required to carry Asn at position 5 and Met or Ile at the equally critical position 9. Inasmuch as Asn and Met are rare residues and Ile, too, is not a common residue, the typical somatic cell type can supply only several hundred host nonapeptides having the high binding affinity toward H-2Db. Under the circumstance, there is no problem in memorizing the selfness of all of them. Accordingly, T epitopes are almost invariably chosen from the high affinity nonapeptides that are present in their viral proteins.