Functional analysis of vocal folds by transcutaneous laryngeal ultrasonography in patients undergoing thyroidectomy.

BACKGROUND: Transcutaneous laryngeal ultrasound (TCLUS) can assess Vocal folds (VF) by subjectively identifying mobility or objectively by calculating vocal fold displacement velocity (VFDV). Optimal diagnostic approach (subjective assessment, VFDV estimation or a combination of both) is unresolved; hence, we conducted this prospective study in patients undergoing thyroidectomy. METHOD: Two anaesthetists performed TCLUS pre- and post-operatively for functional assessment of 200 VFs on 100 patients. Their findings were compared with pre-operative flexible laryngoscope (FL) performed by surgeons and with post-operative C-Mac video laryngoscope (C-Mac VL) by another independent anaesthetist. Correlation between FL and TCLUS findings and inter-rater agreement between TCLUS findings of both anaesthetists was analysed. Decision curve analysis (DCA) was performed to compare clinical benefit of hoarseness, subjective VF movement, VFDV, and combined assessment for detecting disabled VFs. RESULTS: We found good correlation between VF mobility on TCLUS and FL (Spearman's r = 0.93, P < 0.0001) as well as C-Mac VL (Spearman's r = 0.83, P < 0.0001) with excellent inter-rater agreement between both anaesthetists. DCA showed combined assessment to have marginally higher clinical benefit than other diagnostic approaches at intermediate threshold probabilities while its benefit was similar to subjective evaluation at higher threshold probabilities. CONCLUSION: Provided achievement of optimal acoustic window, TCLUS can reliably assess disabled VFs with FL reserved for their confirmation or doubtful cases. Subjective assessment of VF mobility should suffice in most cases with additional VFDV estimation reserved pre-operatively for situations with higher risk of VFs disability, and post-operatively when subjective VF assessment findings are discordant from pre-operative status.

Chronic lymphocytic leukemia cells in a lymph node microenvironment depict molecular signature associated with an aggressive disease.

Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host-tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell-activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eµ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response.

Evaluation of Utility of Invasive Electroencephalography for Definitive Surgery in Patients with Drug-Resistant Epilepsy: A Systematic Review and Meta-Analysis.

When noninvasive tests are unable to define the epileptogenic zone in patients, intracranial electroencephalography (iEEG) is a method of localizing the epileptogenic zone. Compared with noninvasive evaluations, it offers more precise information about patterns of epileptiform activity, which results in useful diagnostic information that supports surgical decision-making. The primary aim of the present study was to assess the utility of iEEG for definitive surgery for patients with drug-resistant epilepsy. Online databases such as PubMed, Medline, Embase, Scopus, Cochrane Library, Web of Science, and IEEE Xplore were searched for MeSH terms and free-text keywords. The ROBINS I (risk of bias in non-randomized studies - of interventions) critical appraisal tool was used for quality assessment. The prevalence from different studies was pooled together using the inverse variance heterogeneity method. Egger's regression analysis and funnel plot were used to evaluate publication bias. The systematic review included 18 studies, and the meta-analysis included 10 studies to estimate the prevalence of seizure freedom (Engel class I) in patients undergoing surgery after iEEG. A total of 526 patients were included in the meta-analysis. The follow-up period ranged from 1 to 10 years. The overall pooled estimate of the prevalence of seizure freedom (Engel class I) for patients undergoing surgery after iEEG was 53% (95% confidence interval, 44%-62%). The results additionally demonstrated that 12 studies had a moderate risk of bias and 6 had a low risk. Future studies are crucial to enhance our understanding of iEEG to guide patient choices and unravel their implications.

Novel therapy for therapy-resistant mantle cell lymphoma: multipronged approach with targeting of hedgehog signaling.

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5-7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.

Lymph node-induced immune tolerance in chronic lymphocytic leukaemia: a role for caveolin-1.

Emerging evidence indicates that the tumour microenvironment (TME) regulates the behaviour of chronic lymphocytic leukaemia (CLL). However, the precise mechanism and molecules involved in this process remain unknown. Gene expression profiles of CLL cells from lymph node (LN), bone marrow (BM) and peripheral blood (PB) indicate overexpression of a tolerogenic signature in CLL cells in lymph nodes (LN-CLL). Based on their role in B cell biology, the progression of CLL, or immune regulation, a few genes of this 83-gene signature were selected for further analyses. We observed a significant correlation between the clinical outcomes and the expression of CAV1 (P = 0·041), FGFR1 isoform 8 (P = 0·032), PTPN6 (P = 0·031) and ZWINT (P < 0·001). CAV1, a molecule involved in the regulation of tumour progression in other cancers, was seven-fold higher in LN-CLL cells compared to BM- and PB-CLL cells. Knockdown of CAV1 expression in CLL cells resulted in significantly decreased migration (P = 0·016) and proliferation (P = 0·04). When CAV1 was knocked down in B and T cell lines, we observed an inability to form immune synapses. Furthermore, CAV1 knockdown in CLL cells impaired their ability to form immune synapses with autologous T lymphocytes and allogeneic, healthy T cells. Subsequent analyses of microarray data showed differential expression of cytoskeletal genes, specifically those involved in actin polymerization. Therefore, we report a novel role for CAV1 in tumour-induced immunosuppression during the progression of CLL.

Efficacy of the three-point cuff palpation technique in preventing endobronchial tube migration during positioning in robotic pelvic surgeries.

Background and Aims: During robotic pelvic surgeries, the shortening of endotracheal tube (ETT) tip-to-carina distance (DTC) during pneumoperitoneum with 45° Trendelenburg position can result in endobronchial tube migration. In the three-point ETT cuff palpation (TPP) technique, maximal ETT cuff distension is felt over the tracheal segment located between the cricoid-thyroid membrane and suprasternal notch, which is likely to provide optimal placement. However, the reproducibility and reliability of the TPP technique in preventing endobronchial tube migration are yet to be evaluated. Hence, we compared three ETT placement techniques: TPP technique, intubation guide mark (IGM) technique and Varshney's formula (VF) for the prevention of endobronchial tube migration during robotic pelvic surgeries. Methods: ETT placement by TPP was compared with IGM and VF techniques in 100 American Society of Anesthesiologists physical class II-III patients, by assessing the serial changes in DTC and incidence of endobronchial tube migration throughout the different phases of pneumoperitoneum and Trendelenburg position using t-test and Chi-square test. Changes in the DTC during various phases were also measured. Results: DTC (mean ± standard deviation) at baseline and during pneumoperitoneum was significantly better in TPP technique (2.80 ± 0.62 cm and 1.96 ± 0.66 cm) as compared to both IGM (2.50 ± 1.27 cm and 1.41 ± 1.29 cm) and VF techniques (1.83 ± 1.13 cm and 0.98 ± 1.18 cm), P < 0.001. During pneumoperitoneum, the mean shortening of DTC was 0.84 ± 0.20 cm, and no endobronchial tube migration was found in TPP technique compared to 20% in IGM and 25% in VF techniques, P < 0.001. Conclusion: TPP is a simple and reliable technique, which provides optimal ETT placement and prevents endobronchial tube migration throughout the different phases of robotic pelvic surgeries.

Acceleration of Wound Healing in Diabetic Rats through Poly Dimethylaminoethyl Acrylate-Hyaluronic Acid Polymeric Hydrogel Impregnated with a Didymocarpus pedicellatus Plant Extract.

Wound is the major health problem associated with skin damages and arises because of various types of topical injuries. Furthermore, wounds in patients with diabetes take a relatively long time to heal. Currently, herbal medicines have been extensively used for wound care and management. Here, we engineered polymeric hybrid hydrogel of dimethylaminoethyl acrylate and hyaluronic acid (pDMAEMA-HA), which was impregnated with a herbal extract of Didymocarpus pedicellatus. The developed polymeric hybrid hydrogel system can be used for effective therapy of incurable wounds. Therefore, the development of D. pedicellatus-impregnated pDMAEMA-HA (pDPi-DMAEMA-HA) hybrid hydrogel was accomplished by the synthesis of pDMAEMA-HA hydrogel via the optimization of various reaction parameters followed by impregnation of herbal drugs D. pedicellatus. The developed hydrogel composite was well characterized via various techniques, and swelling kinetics was performed to analyze the water uptake property. The swelling ratio was found to be 1600% in both types of hydrogels. To evaluate the wound healing of these polymeric hydrogels, the Wistar rats full-thickness excision wound model was utilized. The healing strength of hydrogels was determined using measurement of wound contraction and histopathological study. The results of wound healing by these polymeric hydrogels revealed that animals treated with the pDPi-DMAEMA-HA hybrid hydrogel group were found to have a higher level of wound closure as compared to marketed formulation as well as polymeric hybrid hydrogel. The histopathologic examinations implied that pDPi-DMAEMA-HA hybrid hydrogel and polymeric hybrid hydrogel-treated groups exhibited enhanced cutaneous wound repair as well as high level of cellular repair and maintenance compared to the standard group because of hyaluronic acid roles in various stages of wound repair.

Hedgehog-induced survival of B-cell chronic lymphocytic leukemia cells in a stromal cell microenvironment: a potential new therapeutic target.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell-induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell-induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective down-regulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target.

Differential gene expression in murine large cell B-cell lymphoma metastatic variants.

Previous studies from this laboratory have characterized RAW117-P murine large cell B-cell lymphoma and its in vivo selected highly malignant and liver metastatic RAW117-H10 subline for their biological and biochemical properties. In this study, to understand the molecular basis of low and high metastatic behavior of these variant sublines, we have investigated the molecular phenotypes of these cells using differential display techniques and cDNA array analysis. Differential display analysis indicated a significant difference in expression of several genes between these two metastatic variant lymphoma cells. Further analyses of these cells using microarray showed an increased expression of several genes including uPAR1, CRE-BP1, Chop-10, IGF, insulin-like growth factor-IA, STAT6, Cyclin-D1, Cyclin-E, ERBB-3, Alpha NGF, Kruppel-like factor LKLF, (P)19INK4 in metastatic RAW117-H10 cells compared to parental RAW117-P cells. On the other hand, MIP1beta, CD14 antigen, Cathepsin B and MOD are expressed more in RAW117-P cells compared to RAW117-H10 cells. Differential expression of the selected genes was confirmed using semiquantitative RT-PCR techniques. The combination of plasminogen activator and its receptor and IGF-like growth factors, cell cycle regulatory molecules and transcription factors might provide an ideal environment for RAW117-H10 cells to metastasize to distant organs and colonize. Thus these results identify certain differentially expressed genes that are involved in the metastatic properties of these lymphoma cells and lay foundation for further in depth analyses to use this information to develop therapy for metastatic lymphoma.

ATM, CTLA4, MNDA, and HEM1 in high versus low CD38 expressing B-cell chronic lymphocytic leukemia.

PURPOSE: In B-cell chronic lymphocytic leukemia (CLL), high CD38 expression has been associated with unfavorable clinical course, advanced disease, resistance to therapy, shorter time to first treatment, and shorter survival. However, the genes associated with CLL patient subgroups with high and low CD38 expression and their potential role in disease progression is not known. EXPERIMENTAL DESIGN: To identify the genes associated with the clinical disparity in CLL patients with high versus low CD38 expression, transcriptional profiles were obtained from CLL cells from 39 different patients using oligonucleotide microarray. Gene expression was also compared between CLL cells and B cells from healthy individuals. RESULTS: Gene expression analysis identified 76 differentially expressed genes in CD38 high versus low groups. Out of these genes, HEM1, CTLA4, and MNDA were selected for further studies and their differential expression was confirmed by real-time PCR. HEM1 overexpression was associated with poor outcome, whereas the overexpression of CTLA4 and MNDA was associated with good outcome. Down-regulation of HEM1 expression in patient CLL cells resulted in a significant increase in their susceptibility to fludarabine-mediated killing. In addition, when gene expression patterns in CD38 high and low CLL cells were compared with normal B-cell profiles, ATM expression was found to be significantly lower in CD38 high compared with CD38 low CLL as confirmed by real-time reverse transcription-PCR. CONCLUSIONS: These results identify the possible genes that may be involved in cell proliferation and survival and, thus, determining the clinical behavior of CLL patients expressing high or low CD38.

Molecular basis of aggressive disease in chronic lymphocytic leukemia patients with 11q deletion and trisomy 12 chromosomal abnormalities.

In B-cell chronic lymphocytic leukemia (CLL), Rai stage, immunoglobulin gene mutational status, chromosomal abnormalities, CD38 and ZAP-70 expression were used as prognostic markers. In this study, to understand the molecular basis of chromosomal abnormalities leading to tumor progression, 90 CLL patients were grouped into poor prognosis (with 11q deletion and trisomy 12) and good prognosis (with normal karyotype and 13q deletion) and their clinical outcome was assessed. Gene expression profiles of 35 CLL samples with poor outcome (11q deletion, n=9; trisomy 12, n=5) and good outcome (13q deletion, n=13; normal karyotype, n=8) were analyzed using oligonucleotide microarray. Significance analysis of microarray (SAM) identified 27 differentially expressed genes between these two subgroups with significant overexpression of ATF5 and underexpression of CDC16, PCDH8, SLAM, MNDA and ATF2 in CLL patients with poor outcome. ATF5 gene expression in CLL was further studied because of its role in the regulation of cell cycle progression/differentiation and apoptosis. The overexpression of ATF5 was confirmed by real-time PCR using 39 CLL samples from the poor and good outcome groups. ATF5 was significantly (p<0.001) overexpressed in the poor outcome group. Furthermore, ATF5 expression was significantly higher in the 11q deletion as well as trisomy 12 group alone compared to the 13q deletion and normal karyotype groups. ATF5 overexpression was also associated with significantly (p=0.04) shorter time to treatment. Similarly, expression of five underexpressed genes also correlated with longer time to treatment. Thus, this report demonstrates that ATF5 may be one of the key genes involved in increased proliferation and survival in 11q deletion or trisomy 12, whereas CD16, CD86, SLAM, MNDA and ATF2 may be involved in the decreased proliferation of CLL cells with 13q deletion or normal karyotype.

Bioinspired Nanotheranostic Agents: Synthesis, Surface Functionalization, and Antioxidant Potential.

Bioinspired synthesis of nanomaterials is highly advantageous as a natural and cost-effective resource. Development of noble metal nanotheranostic agents was achieved through bioinspired synthetic routes. These biosynthesized nanoparticles were characterized by various analytical techniques including absorption spectroscopy, FTIR and electron microscopy (SEM and TEM). A large number of medicinal plants were screened, among which Potentilla fulgens (PF, vajradanti) and Camellia sinensis (CS, green tea) were found to produce nanomaterials with higher yields. Plant (PF and CS) mediated metallic nanoparticles had added advantage of metal reduction and simultaneous phytochemical capping over chemically synthesized procedures, which require multiple reagents. Antioxidant potential of the nanomaterials was determined by in vitro antioxidant assays confirming substantial antioxidant properties, which was due to the presence of phytochemicals on the nanoparticle surface. Flavonoids and catechins on the nanomaterial surface served as the supplier of hydroxyl groups for further derivatization. The surface of the nanoparticles was engineered by conjugating imaging and therapeutic moieties, resulting in the formation of theranostic nanoagents. The multimodal agents were characterized and the extent of drug loading was determined to validate the efficacy of those nanoconjugates. These bioinspired multimodal nanoprobes can serve as essential diagnostic and therapeutic tools in ongoing biomedical research.

Role of CTLA4 in the proliferation and survival of chronic lymphocytic leukemia.

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, (3)H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.

Candidate tumor suppressor and pVHL partner Jade-1 binds and inhibits AKT in renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor pVHL is lost in the majority of clear-cell renal cell carcinomas (RCC). Activation of the PI3K/AKT/mTOR pathway is also common in RCC, with PTEN loss occurring in approximately 30% of the cases, but other mechanisms responsible for activating AKT at a wider level in this setting are undefined. Plant homeodomain protein Jade-1 (PHF17) is a candidate renal tumor suppressor stabilized by pVHL. Here, using kinase arrays, we identified phospho-AKT1 as an important target of Jade-1. Overexpressing or silencing Jade-1 in RCC cells increased or decreased levels of endogenous phospho-AKT/AKT1. Furthermore, reintroducing pVHL into RCC cells increased endogenous Jade-1 and suppressed endogenous levels of phospho-AKT, which colocalized with and bound to Jade-1. The N-terminus of Jade-1 bound both the catalytic domain and the C-terminal regulatory tail of AKT, suggesting a mechanism through which Jade-1 inhibited AKT kinase activity. Intriguingly, RCC precursor cells where Jade-1 was silenced exhibited an increased capacity for AKT-dependent anchorage-independent growth, in support of a tumor suppressor function for Jade-1 in RCC. In support of this concept, an in silico expression analysis suggested that reduced Jade-1 expression is a poor prognostic factor in clear-cell RCC that is associated with activation of an AKT1 target gene signature. Taken together, our results identify 2 mechanisms for Jade-1 fine control of AKT/AKT1 in RCC, through loss of pVHL, which decreases Jade-1 protein, or through attenuation in Jade-1 expression. These findings help explain the pathologic cooperativity in clear-cell RCC between PTEN inactivation and pVHL loss, which leads to decreased Jade-1 levels that superactivate AKT. In addition, they prompt further investigation of Jade-1 as a candidate biomarker and tumor suppressor in clear-cell RCC.

Tumor-specific peptide-based vaccines containing the conformationally biased, response-selective C5a agonists EP54 and EP67 protect against aggressive large B cell lymphoma in a syngeneic murine model.

Vaccines to large B cell lymphoma were made by the covalent attachment of an epitope from the gp70 glycoprotein (SSWDFITV) to the N-termini of the conformationally biased, response-selective C5a agonists EP54 (YSFKPMPLaR) and EP67 (YSFKDMP(MeL)aR). Syngeneic Balb/c mice were immunized with these EP54/EP67-containing vaccines and challenged with a lethal dose of the highly liver metastatic and gp70-expressing lymphoma cell line RAW117-H10 to evaluate the ability of these vaccines to induce protective immune outcomes. All mice immunized with SSWDFITVRRYSFKPMPLaR (Vaccine 2) and SSWDFITVRRYSFKDMP(MeL)aR (Vaccine 3) were protected to a lethal challenge of RAW117-H10 lymphoma (>170 days survival) and exhibited no lymphoma infiltration or solid tumor nodules in the liver relative to unvaccinated controls (<18 days survival). Vaccines 2 and 3 contained the protease-sensitive double-Arg (RR) linker sequence between the epitope and the EP54/EP67 moieties in order to provide a site for intracellular proteases to separate the epitope from the EP54/EP67 moieties once internalized by the APC and, consequently, enhance epitope presentation in the context of MHC I/II. These protected mice exhibited an immune outcome consistent with increased involvement of CD8(+) and/or CD4(+) T lymphocytes relative to controls and mice that did not survive or showed low survival rates as with Vaccines 1 and 4, which lacked the RR linker sequence. CD8(+) T lymphocytes activated in response to Vaccines 2 and 3 express cytotoxic specificity for gp70-expressing RAW117-H10 lymphoma cells, but not antigen-irrelevant MDA-MB231A human breast cancer cells. Results are discussed against the backdrop of the ability of EP54/EP67 to selectively target antigens to and activate C5a receptor-bearing antigen presenting cells and the prospects of using such vaccines therapeutically against lymphoma and other cancers.