Complementary activation of peripheral natural killer cell immunity in nasopharyngeal carcinoma

NK cells and αβ- and γδ-CTL play important roles in cellular immunity against tumors. We previously demonstrated that NPC patients have a quantitative and qualitative deficit in γδ-CTL and EBV-specific αβ-CTL when compared to normal subjects and NPC long-term survivors. In this study we report further observations of a complementary activation of peripheral NK cells in NPC patients. The NK cells in these patients, compared to those of healthy subjects and NPC survivors, were preferentially activated in response to the stimulation of myeloma cell line XG-7 and expanded in the presence of exogenous IL-2. The production of IFN-γ was lowest in the patient group, whereas IL-12, IL-15 and TNF-α were produced in higher levels in patients than in the donors and survivors. The cytolytic effect of the NK cells against NPC cells in the patient group was also higher than that of the donors and survivors. Furthermore, the patients at later stages of NPC had lower γδ-CTL activity but higher NK cytotoxicity towards NPC targets, with higher production of IL-12, IL-15 and TNF-α but lower production of IFN-γ than in patients at earlier stages. This might be part of a triggered compensatory re-activation of the innate immunity, believed to be mediated through various cytokines and chemokines when adaptive T cell immunity is breached. Together, these data suggest complementary roles of innate and adaptive immune response in tumor immunity where NK cells, γδ- and αβ-CTL compensate for the deficits of one another at different stages of tumor invasion. © 2006 Japanese Cancer Association.published_or_final_versio

Phenotypic Detection of Clonotypic B Cells in Multiple Myeloma by Specific Immunoglobulin Ligands Reveals their Rarity in Multiple Myeloma

In multiple myeloma, circulating “clonotypic” B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential “feeder” cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10−3, this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology

Evaluation of vardenafil for the treatment of subjective tinnitus: a controlled pilot study

<p>Abstract</p> <p>Background</p> <p>Vardenafil (Levitra^®) represents a potent and highly selective phosphodiesterase type 5 (PDE5) inhibitor, which is established for treatment of various diseases. There are several unpublished reports from patients stating that vardenafil has a considerable therapeutic effect on their concomitant tinnitus. This pilot study was conducted to specifically assess the effect of vardenafil in patients with chronic tinnitus.</p> <p>Methods</p> <p>This trial was based on a prospective, randomized, double-blind, placebo-controlled, parallel group design. Fourty-two consecutive subjects with mon- or binaural chronic tinnitus received 10 mg vardenafil (N = 21) or matching placebo tablets (N = 21) administered orally twice a day over a period of 12 weeks. Clinical examination and data acquisition took place at each visit: at baseline, after 4 weeks, after 12 weeks (end of treatment with study medication), and at non-medicated follow-up after 16 weeks. Assessment of clinical effectiveness was based on a standardized tinnitus questionnaire (TQ), the Short Form 36 health survey (SF-36), audiometric measurements (mode, pitch and loudness of tinnitus; auditory thresholds) and biomarkers of oxidative stress in patients' blood (malondialdehyde, protein carbonyl, homocysteine and total antioxidative status). Therapeutic efficacy was evaluated by comparison of subjective and objective parameters with baseline data between both treatment groups (ANCOVA).</p> <p>Results</p> <p>Vardenafil had no superior efficacy over placebo in the treatment of chronic tinnitus during this study. The primary efficacy criterion 'TQ total score' failed to demonstrate significant improvement compared to placebo. Subjective reports of TQ subscales and general quality of life areas (SF-36), objective audiometric examinations as well as investigated biomarkers for oxidative stress did not reveal any significant treatment effects. The safety profile was favorable and consistent with that in other vardenafil studies.</p> <p>Conclusion</p> <p>Although hypoxia and ischemia play a special role in the pathogenesis of tinnitus, the PDE5-inhibitor-induced increase of nitric oxide-mediated vasodilatation exerted no specific influence on tinnitus symptomatology. Considering the unclear risk of rarely associated hearing impairment, systemic application of vardenafil or other PDE5 inhibitors prove to be not appropriate for therapy of chronic tinnitus.</p

p53 Gene Repair with Zinc Finger Nucleases Optimised by Yeast 1-Hybrid and Validated by Solexa Sequencing

The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation ‘hotspots’. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci

Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells

Multiple myeloma (MM) accounts for 1 % of all cancer deaths. Although treated aggressively, almost all myelomas eventually recur and become resistant to treatment. Atiprimod (2-(3-Diethylaminopropyl)-8,8-dipropyl-2-azaspiro[4,5] decane dimaleate) has exerted anti-inflammatory activities and inhibited oeteoclast-induced bone resorption in animal models and been well tolerated in patients with rheumatoid arthritis in phase I clinical trials. Therefore, we investigated its activity in MM cells and its mechanism of action. We found that Atiprimod inhibited proliferation of the myeloma cell lines U266-B1, OCI-MY5, MM-1, and MM-1R in a time- and dose-dependent manner. Atiprimod blocked U266-B1 myeloma cells in the G0/G1 phase, preventing cell cycle progression. Furthermore, Atiprimod inhibited signal transducer and activator of transcription (STAT) 3 activation, blocking the signalling pathway of interleukin-6, which contributes to myeloma cell proliferation and survival, and downregulated the antiapoptotic proteins Bcl-2, Bcl-XL, and Mcl-1. Incubation of U266-B1 myeloma cells with Atiprimod induced apoptosis through the activation of caspase 3 and subsequent cleavage of the DNA repair enzyme poly(adenosine diphosphate-ribose) polymerase. Finally, Atiprimod suppressed myeloma colony-forming cell proliferation in fresh marrow cells from five patients with newly diagnosed MM in a dose-dependent fashion. These data suggest that Atiprimod has a role in future therapies for MM