Lytic Units Research Articles

Effect Of NKG2D And DNAM-1 Expression On CD56dim NK Cell And Cytotoxic Activity During Training

PURPOSE: Natural killer (NK) cell cytotoxic activity is controlled by both activating and inhibitory receptors. NKG2D and DNAM-1 are activating receptors and are responsible for eliminating tumor and virus-infected cells. We previously reported that chronic exercise induced changes in cytolytic activity per NK cell. In the present study, we examined the relationships between NK cell cytotoxicity and expression of NKG2D and DNAM-1 on CD56dim NK cells during training. METHODS: 8 college-level male wrestlers underwent 1-month of pre-season training. Drills were performed 4 hours/day, 6 days/week. 3 morning resting blood samples were taken pre-training (PRE), 1 day after the end of training (END) and 5 days after training (POST). Phenotype and receptor density of NK cells were analyzed by flow cytometry. Cytotoxicity was measured using a standard 51Cr release assay, with effecter to target cell ratios of 20:1, 10:1, 5:1 and 2.5:1. The lytic unit was calculated as an index of per-cell cytotoxicity. Linear regression analyses were applied to determine the relationship between cytotoxicity and the expression of activating receptors. RESULTS: There were no changes in circulating CD56dim and CD56bright NK cell ratios, or in cytolytic activity (20:1) throughout the experiment. Per-cell cytotoxicity showed large inter-individual variations but did not change significantly. The expression of NKG2D increased (p<0.001) at END (p<0.001) and POST (p=0.004). In contrast, DNAM-1 expression did not change. Cytotoxicity per NK cell correlated positively with the expression of NKG2D (r=0.518, p=0.010), but no significant relationship was established between cytotoxicity and DNAM-1 expression. DISCUSSION: The impact of training in this study seemed less than that observed in our previous study, because 7 of the 8 subjects showed no decrease in cytotoxic activity per NK cell at END. Nevertheless, analysis of the relationship with activating receptors indicated that NKG2D expression, and not DNAM-1 expression, affects the cytotoxicity per NK cell. CONCLUSIONS: The results suggest that under resting conditions, cytotoxic activity per NK cell is regulated by NKG2D activating receptors, and not by DNAM-1 receptors. Supported by the Grant-in-Aid for Scientific Research B, 21300257, Japan Society for the Promotion of Science.

Abstract 5609: Resveratrol as a modulator of human tumor proliferation and immunologic sensitivity

Abstract Integrative cancer treatment employing natural products (NPs) in conjunction with conventional therapeutic modalities (Tx) is attractive for tumors that are difficult to control clinically. In this context, pluripotent activities of NPs have the potential of affecting multiple processes relevant to cancer growth. Unfortunately, there is little empiric evidence to elucidate clinically useful effects of NPs making it difficult to optimize Tx regimens. This challenge justifies translational studies to characterize NP effects on processes relevant to tumor control. In the current study, we assessed the action of Resveratrol (RV), a phytoalexin present in the skin of red grapes on the sensitivity of human tumors to proliferation inhibition and antitumor immunity. Proliferation was assessed with MTS assay; immune-mediated lysis with 51Chromium release assay; and gene expression with real time PCR. Using the following ATCC cell lines: malignant melanoma (MM); renal (RC); ovarian (OC); non-small cell lung (NSCLC); pancreatic (PC); breast (BC); and bladder (BLC) cancer, significant dose-dependent proliferation inhibition (paired, 2-tail t tests) in RV-treated cells was demonstrated for all tumors with rank order of sensitivity being: MM &amp;gt; RC &amp;gt; OC &amp;gt; NSCLC &amp;gt; BC &amp;gt; BLC &amp;gt; PC. Significant inhibition (p &amp;lt; 0.05) was also observed with tumor cells from surgical specimens from patients with colon, RC, PC, and OC. The substantial proliferation inhibitory effects of RV on MM (&amp;gt; 80% at 10μg/mL; p = 0.005) and RC (&amp;gt; 65% at 10μg/mL; p = 0.02) was associated with significant chanages in quantitative expression of various pro and anti-apoptotic regulatory genes. For example, RV-treated MM showed &amp;gt; 8 fold down-regulation of anti-apoptotic BCL2 in association with &amp;gt; 13 fold increase in the death associated kinase, DPK1 and RV-treated RC showed 2-3 fold up-regulation of caspases 1, 5, 7, 10, and 14. Moreover, these studies also revealed significantly increased (2-4 fold) expression of different TNF and TRAIL-related death receptors in RV-treated RC and MM cells. Subsequently, testing the effects of RV-pretreated RC and MM to different forms of antitumor immunity showed: 1)RV pre-treated RC are significantly more sensitive to lysis by IL2-activated human lymphocytes compared to control cells (270 vs 121 lytic units respectively, p=0.04); and, 2) proliferation of MM is inhibited significantly (p&amp;lt;0.05) in an additive fashion by the combination of RV + rTNF or RV + rTRAIL. This study demonstrates that RV can favorably modulate tumor cell proliferation and sensitivity to tumor immune mechanisms in some of the most difficult to treat human cancer histologies. These sorts of studies should facilitate development of integrative treatment modalities to exploit the pluripotent activities of NPs such as RV in specific oncologic settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5609.

Enhancement of natural killer cell activity of aged mice by modified arabinoxylan rice bran (MGN-3/Biobran)

The present study is aimed to examine the possibility of enhancement of natural killer (NK) cell activity in aged C57BL/6 and C3H mice using MGN-3, a modified arabinoxylan from rice bran. Intraperitoneal injection of MGN-3 (10 mg kg(-1) per day) caused a remarkable increase in the peritoneal NK activity as early as 2 days (35.2 lytic units), and the level remained elevated through day 14. The control aged mice had a level of 5.8 lytic units. Enhancement in NK activity was associated with an increase in both the binding capacity of NK cells to tumour targets and in the granular content as measured by BLT-esterase activity. Treatment did not alter the percentage of peritoneal NK cells. Data showed that peritoneal macrophages inhibit NK activity. In conclusion, MGN-3 enhances murine NK activity of aged mice and may be useful for enhancing NK function in aged humans.

ORIGINAL ARTICLE: Suppression of Natural Killer Cell Cytotoxicity in Postpartum Women

Citation Groer M, El‐Badri N, Djeu J, Harrington M, Van Eepoel J. Suppression of natural killer cell cytotoxicity in postpartum women. Am J Reprod Immunol 2010; 63: 209–213Problem Natural Killer (NK) cell numbers and cytotoxicity are suppressed during pregnancy. Little is known about postpartum NK number and function.Method of study Postpartum women (n = 39) were studied at one week and then monthly over the first six postpartum months. The standard natural killer cell cytotoxicity assay (NKCA) was performed. This is a Cr51 release assay from K562 cells cultured with peripheral blood mononuclear cells (PBMCs).Results Data indicate suppression of NK cytotoxicity in postpartum women. Cytotoxicity at each effector:target (E:T) ratio showed a drop from 1 week postpartum, reaching a nadir at around 2 months, and a trend towards recovery of cytotoxicity from 3 to 6 months. Lytic units (LUs) from pre‐incubated cells from postpartum women were lower than age‐matched, non‐pregnant, non‐postpartum controls through the fifth postpartum month.Conclusion These data suggest that the postpartum period, like pregnancy, is characterized by decreased NK cytotoxicity activity. This suppressed NK cytotoxic effect may result as a response to interaction with tolerized fetal microchimeric cells accumulated during pregnancy in maternal blood and tissues.

Extremely Low Frequency-Magnetic Fields (ELF-EMF) occupational exposure and natural killer activity in peripheral blood lymphocytes

Extremely Low Frequency-Magnetic Fields (ELF-MF) are possible carcinogens to humans and some data suggest that they can act as promoters or progressors. Since NK cells play a major role in the control of cancer development, an adverse effect on ELF-MF on NK function has been hypothesized. We examined NK activity in 52 workers exposed to different levels of ELF-MF in various activities. Individual exposure was monitored during 3 complete work-shifts using personal dosimeters. Environmental exposure was also monitored. ELF-MF levels in the workers were expressed as Time-Weighted Average (TWA) values. NK activity was measured in peripheral blood lymphocytes (PBL). In the whole group the median occupational TWA was 0.21 µT. According to the TWA levels, workers were classified as low exposed (26 subjects, TWA ≤ 0.2 µT) and higher exposed workers (26 subjects; TWA > 0.2 µT). In higher exposed workers, we observed a trend to reduce NK activity compared to low exposed, but the difference was not significant. Then we selected a subgroup of highest exposed workers (12 subjects; TWA > 1 µT); no difference was observed between low and highest exposed subjects in the main personal variables. Considering both E: T ratios from 12:1 to 50:1 and Lytic Units, a significant reduction in NK activity was observed in the highest exposed workers compared to the low exposed. Multivariate analysis showed a significant negative correlation between exposure and LU, while no correlation was evidenced with other personal characteristics. ELF-MF are considered possible carcinogens, and existing data suggest that they can act as promoters. Due to the role of NK activity in host defence against cancer, the results obtained in this study in workers exposed to ELF-MF levels exceeding 1 µT are in agreement with this hypothesis, and support the need for further investigation in this field.

Natural killer cell activity during measles

Natural killer cells are postulated to play an important role in host anti-viral defences. We measured natural killer cell activity in 30 individuals with acute measles (73 +/- 21 lytic units (LU)/10(7) cells) and 16 individuals with other infectious diseases (149 +/- 95 LU) and found it reduced compared with values for adults (375 +/- 70 LU; P less than 0.001) or children (300 +/- 73 LU, P less than 0.01) without infection. Reduced natural killer cell activity was found in measles patients with (84 +/- 30 LU) and without (55 +/- 18 LU) complications and was present for at least 3 weeks after the onset of the rash. Activity was increased by in vitro exposure of cells to interleukin-2. Depressed natural killer cell activity parallels in time the suppression of other parameters of cell-mediated immunity that occurs during measles.

Long-term cultures of human peripheral blood lymphocytes with recombinant human interleukin-2 generate a population of virtually pure CD3+CD16-CD56- large granular lymphocyte LAK cells

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.

Gemcitabine enhances the sensitivity of renal cell cancer to cytolysis by IL2-activated lymphocytes

14020 Background: Limited clinical efficacy of immunotherapy (IT) necessitates new treatment (Tx) strategies. A relatively novel approach that is clinically feasible exploits the capacity of chemotherapy to affect tumor functions that govern sensitivity to immune-mediated killing. The current study tested this with renal cell cancer (RCC) cells exposed to clinically achievable doses of drugs prior to assessing their sensitivity to cytolysis by IL2-activated human lymphocytes (IL2 Ly). Methods: Human RCC line 769-P was incubated with pharmacologic concentrations of vinblastine, cisplatinum, paclitaxel, or gemcitabine (Gz) prior to evaluating killing by IL2 Ly. Optimum results with Gz prompted feasibility and mechanistic studies. Results: In vitro exposure to 20 uM Gz for 4 hrs, which is ≤ levels achieved in patients receiving Gz Tx of 1 gm/M2; 30 min infusion (½ life=60 min) increased RCC lysis by IL2 Ly from multiple donors from 623 ± 370 to 780 ± 477 lytic units (media vs Gz-precultured cells respectively, p=0.008). Genomic analysis demonstrated increased lytic sensitivity was associated with increased mRNA expression for: (i) adhesion molecules, ICAM-1, Necl-2 and MCAM (6.5, 2 and 2 fold respectively); (ii) MHC Class I-related molecules, MR-1 (7 fold) and HLA-A, B, E and F (1.5–3 fold); and, (iii) death receptors, FAS and TRAIL-receptor 2 (2 fold respectively). Flow cytometry confirmed the 2 fold increase in FAS death receptors on GZ-treated RCC cells without changes in tumor expression of killer cell inhibitory ligands associated with HLA-A, B, and C molecules. Gz suppressed cytotoxic function generation in Ly cultured for 3 days with IL2 but was not suppressive in 6 day cultures. Improved survival was shown in RCC-bearing immunocompetent mice given Gz + IL2 compared to mice treated with either drug alone. Conclusions: The results demonstrate RCC cells that survive Tx with a pharmacological dose of Gz exhibit significantly increased sensitivity to killing by human IL2 Ly. These methods can be used to identify classes of drugs that might be exploited to enhance sensitivity of chemo-resistant human tumors to immunotherapy. No significant financial relationships to disclose.

Granzyme B-induced Cell Death Involves Induction of p53 Tumor Suppressor Gene and Its Activation in Tumor Target Cells

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.

Effects of a Preincisional 14-Day Course of Valerian on Natural Killer Cell Activity in Sprague-Dawley Male Rats Undergoing Abdominal Surgery

The American Cancer Society estimated that more than 1 million new cancer cases were diagnosed in 2005 and a majority of these patients died from metastatic spread. The standard for treating solid tumor cancer is surgical resection. However, it has been suggested that surgical resection may, in fact, promote metastasis. One of the body's natural defenses to combat metastasis is the activity of natural killer (NK) cells. NK cells serve as a vital mediator of detection during the early innate immune response and destruction of aberrant cells. It has been demonstrated that benzodiazepines may ameliorate surgery-induced suppression of NK cell activity. We examined the effect of a 14-day course of valerian, a herbal anxiolytic, on NK cell activity in Sprague-Dawley rats. Thirty-five rats were assigned to 1 of 3 groups: (1) surgical animals administered research grade valerian, 15 mg/kg solubilized in peanut oil; (2) surgical animals administered peanut oil (vehicle); and (3) anesthesia-only animals administered valerian. One day before the 14-day course of valerian, blood was drawn to assay baseline NK cell activity. On experimental day, all animals were administered isoflurane anesthesia. Surgical animals underwent a standard laparotomy whereas anesthesia-only rats were anesthetized for the same period of time as the surgical rats. Twenty-four hours postexperiment animals underwent a second blood draw to assay NK cell activity. Analysis of covariance (ANCOVA) was used to analyze NK cell activity (measured in lytic units). Our results suggested that there was no difference (P = .9) in suppression within or between groups. Clinical studies with valerian have been published but with small numbers and some ambiguity. Further research regarding valerian's effectiveness as a modulator of NK cell activity and whether dosage or route of administration is a factor in modulation is still warranted.

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Association of Resolution of Major Depression With Increased Natural Killer Cell Activity Among HIV-Seropositive Women

Depression is a potential risk factor for morbidity and mortality among patients with numerous medical conditions, including HIV disease, and it is also associated with decrements in immune function, such as natural killer (NK) cell activity. This study examined whether improvements in the diagnostic status of major depression are related to increases in NK cell activity among HIV-seropositive women. HIV-seropositive women were recruited as part of a longitudinal cohort study and underwent comprehensive medical and psychiatric evaluations during a 2-year period. Fifty-seven women had complete NK cell activity and depression data measured at two time points and were examined for associations between changes in depression status and alterations in NK cell activity over time. Among the 57 HIV-seropositive women, improvements in the diagnostic status of depression and decreases in scores on the 17-item Hamilton Depression Rating Scale were significantly associated with increases in NK cell activity over time, as measured in lytic units. Eleven women (19.3%) had a major depression diagnosis that resolved over time, and this group also had a significant increase in cell activity measured in lytic units during this period. This study suggests that depression may impair certain aspects of innate cellular immunity relevant to delaying the progression of HIV disease and that these alterations are reversible with the resolution of a depressive episode. These findings support an examination of NK cell activity in assessments of the relationship between depression and morbidity and mortality in HIV disease.

Impaired cytolytic activity in peripheral blood T cells from renal cell carcinoma patients

Classic T lymphocyte cytotoxicity is mediated through the T cell receptor (TCR). Defects in TCR signal transduction and cytolytic activity have been reported in tumor infiltrating T lymphocytes. We hypothesized that impaired cytotoxicity occurs in peripheral blood T cells from renal cell carcinoma (RCC) that can be reversed by exposure to rhIL-2. Peripheral blood mononuclear cells (PBMC) from 29 RCC patients and 29 healthy volunteers were isolated and cultured in the absence or presence of 10 IU/ml rhIL-2. A redirected cytotoxicity assay that requires TCR signal transduction was used with chromium-labeled P815 target cells, effector PBMC and anti-CD3 antibody. Target cell lysis was measured in “lytic units” (LU). Mean LU from RCC patients was lower than that of healthy volunteers (105.8 LU vs. 194.6 LU, P = 0.025). Exposure to rhIL-2 increased T cell-mediated lysis in both groups. Disruption of T cell cytotoxicity in RCC patients can be overcome by exposure to rhIL-2.

Changes In The Proportion Of Cd56dim And Cd56bright Natural Killer Cells During Incremental Exercise

Our previous research has shown changes in the proportions of natural killer (NK) cell subsets with different cy to toxic ities during one month of intensive training. These changes could partly explain an observed decrease in lytic units per NK cell at the end of training. There has been no report on changes in NK cell subset proportions during an acute bout of exercise. Researcher indicated that the increase in NK cell cytolytic activity following vigorous exercise was less than that expected from the associated increase in NK cell counts. PURPOSE We investigated the cell counts and the relative proportions of CD3-CD16brightCD56dim (CD56dim) and CD3-CD16dim/-CD56bright (CD56bright) NK cells during an incremental exercise to test the hypothesis that high-intensity exercise induces an increase in the proportion of low cytotoxic NK cells in the peripheral blood. METHODS Nine male students exercised on a cycle ergometer for 5 min at 50, 90, 120 and 140% ventilatory threshold (VT)-VO. Blood samples were taken at rest and at the end of each exercise-intensity stage. The phenotypes of circulating NK cells were determined by flow cytometry. RESULTS Both CD56dim (cells with high cytotoxicity, p<0.001) and CD56bright (cells with low cytotoxicity, p<0.02) cell counts increased exponentially as exercise intensity increased. Total NK cell cytolytic activity followed a corresponding pattern (p<0.001). The proportion of CD56dim NK cell expressed as a percentage of total lymphocyte counts increased significantly (p<0.001). In contrast, the percentage of CD56bright NK cells remained unchanged throughout the experiments. CONCLUSIONS These results suggest that a bout of high-intensity exercise did not induce an increase in the proportion of the CD56bright NK cell subset with low cytolytic activities. Supported by Meiji Life Insurance Company.

Randomized controlled trial of exercise and blood immune function in postmenopausal breast cancer survivors

The objective was to determine the effects of exercise training on changes in blood immune function in postmenopausal breast cancer survivors. Fifty-three postmenopausal breast cancer survivors were randomly assigned to an exercise (n=25) or control group (n=28). The exercise group trained on cycle ergometers three times per week for 15 wk. The control group did not train. The primary end point was change in natural killer cell cytotoxic activity in isolated peripheral blood mononuclear cells. Secondary end points were changes in standard hematological variables, whole blood neutrophil function, the phenotypes of isolated mononuclear cells, estimations of unstimulated and phytohemaglutinin-stimulated mononuclear cell function (rate of [3H]thymidine uptake), and the production of proinflammatory [interleukin (IL)-1alpha, tumor necrosis factor-alpha, IL-6] and anti-inflammatory cytokines (IL-4, IL-10, transforming growth factor-beta1). Statistical tests were two-sided (alpha <0.05). Fifty-two participants completed the trial. Intention-to-treat analyses, which included the baseline value as a covariate, showed significant differences between groups for change in percent specific lysis of a target natural killer cell at all five effector-to-target ratios (adjusted mean between-group change over all 5 effector-to-target ratios = +6.34%; P <0.05 for all comparisons), the lytic activity per cell (adjusted mean between-group change = -2.72 lytic units; P=0.035), and unstimulated [3H]thymidine uptake by peripheral blood lymphocytes (adjusted mean between-group change = +218 per dpm x 10(6) cells; P = 0.007). There were no significant differences between groups for change in any other end point. Exercise training increased natural killer cell cytotoxic activity and unstimulated [3H]thymidine uptake by peripheral blood lymphocytes in postmenopausal breast cancer survivors.

De Novo Generation of Cationic Antimicrobial Peptides: Influence of Length and Tryptophan Substitution on Antimicrobial Activity

Comparison of human immunodeficiency virus lentiviral lytic peptide 1 with other host-derived peptides indicates that antimicrobial properties of membrane-active peptides are markedly influenced by their cationic, hydrophobic, and amphipathic properties. Many common themes, such as Arg composition of the cationic face of an amphipathic helix and the importance of maintaining the hydrophobic face, have been deduced from these observations. These studies suggest that a peptide with these structural properties can be derived de novo by using only a few strategically positioned amino acids. However, the effects of length and helicity on antimicrobial activity and selectivity have not been objectively evaluated in the context of this motif. To address these structure-function issues, multimers of a 12-residue lytic base unit (LBU) peptide composed only of Arg and Val residues aligned to form idealized amphipathic helices were designed. Bacterial killing assays and circular dichroism analyses reveal a strong correlation between antibacterial activity, peptide length, and propensity to form a helix in solvent mimicking the environment of a membrane. Increasing peptide length beyond two LBUs (24-residue peptides) resulted in no appreciable increase in antimicrobial activity. Derivatives (WLBU) of the LBU series were further engineered by substituting Trp residues in the hydrophobic domains. The 24-residue WLBU2 peptide was active at physiologic NaCl concentrations against Staphylococcus aureus and mucoid and nonmucoid strains of Pseudomonas aeruginosa. Further, WLBU2 displayed the highest antibacterial selectivity of all peptides evaluated in the present study by using a coculture model of P. aeruginosa and primary human skin fibroblasts. These findings provide fundamental information toward the de novo design of an antimicrobial peptide useful for the management of infectious diseases.

TLR9-targeted CpG immunostimulatory treatment of metastatic melanoma: A phase II trial with CpG 7909 (ProMune)

7513 Background: Malignant melanoma is widely accepted as an immunologically responsive tumor, but large recently reported chemobiotherapy trials have been disappointing and prognosis is poor. The new synthetic oligodeoxynucleotide CpG 7909 activates plasmacytoid dendritic cells (pDC) and B cells through specific interaction with Toll-like receptor 9 (TLR9) and is a strong activator of both innate and specific immunity. CPG 7909 crossreacts with mouse TLR9 and has shown impressive antitumor activity in preclinical tumor models when used as monotherapy. Methods: Patients with confirmed metastatic melanoma (stage IV without CNS metastases) were enrolled into an open-label, multicenter, single arm study. Pts received 6 mg CPG 7909 weekly by SC injection for 24 weeks or until disease progression in an outpatient setting. Disease status was assessed at screening and weeks 8, 16, and 24 according to RECIST. Results: 20 pts (5 female, 15 male, aged between 37 and 74) were enrolled. Two pts achieved a confirmed partial response (PR) and one has maintained this response with continuing therapy for over 13 months. Three pts achieved stable disease (SD). CPG 7909 was well tolerated. Adverse events included transient injection site reactions (erythema, swelling, induration), fever and arthralgias. Hematological and non-hematological toxicities were limited, transient and did not result in any withdrawals. Phenotyping of PBMC revealed activation of pDC under CPG 7909 therapy consistent with the mode of action and pts exhibiting PR or SD could be distinguished from non-R pts by differential dynamics in NK cell-cytotoxicity (1.6-fold increase vs. 1.7-fold decrease in lytic units, p<0.05) during the first 8 weeks of treatment. Conclusions: CPG 7909 exerts anti-tumor activity in pts with metastatic melanoma, can be administered safely and induces a phenotypic signature in PBMC associated with exposure and, possibly, response to therapy. A randomised phase II/III trial has been initiated to compare efficacy and safety of two dose levels of CPG 7909, CPG 7909 in combination with DTIC, and DTIC alone. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group

Cellular and humoral immune abnormalities in Gulf War veterans.

We examined 100 symptomatic Gulf War veterans (patients) and 100 controls for immunologic assays. The veterans and controls were compared for the percentage of T cells (CD3); B cells (CD19); helper:suppressor (CD4:CD8) ratio; natural killer (NK) cell activity; mitogenic response to phytohemagglutin (PHA) and pokeweed mitogen (PWM); level of immune complexes; myelin basic protein (MBP) and striated and smooth muscle autoantibodies; and antibodies against Epstein-Barr virus, cytomegalovirus, herpes simplex virus type 1 (HSV-1), HSV-2, human herpes Type 6 (HHV-6), and Varicella zoster virus (VZV). The percentage of T cells in patients versus controls was not significantly different, whereas a significantly higher proportion of patients had elevated T cells compared with controls. The percentage of B cells was significantly elevated in the patients versus the controls. The NK cell (NK) activity was significantly decreased in the patients (24.8 +/- 16.5 lytic units) versus the controls (37.3 +/- 26.4 lytic units). The percentage of patients with lower than normal response to PHA and PWM was significantly different from controls. Immune complexes were significantly increased in the patients (53.1 +/- 18.6, mean +/- SD) versus controls (34.6 +/- 14.3). Autoantibody titers directed against MBP and striated or smooth muscle were significantly greater in patients versus controls. Finally, the patients had significantly greater titers of antibodies to the viruses compared with the controls (p < 0.001). These immune alterations were detected 2-8 years after participation in the Gulf War. The immune alterations are consistent with exposure to different environmental factors. We conclude that Gulf War syndrome is a multifaceted illness with immune function alterations that may be induced by various factors and are probably associated with chronic fatigue syndrome.

Changes in Plasma Glutamine Concentration and Natural Killer Cell Cytotoxicity During Intensive Training

1760 Our previous research showed decreased lytic units per natural killer (NK) cell after one month of intensive training. Although the proportion of NK cells with a low cytotoxicity was increased, this change did not entirely explain the observed changes. PURPOSE: We thus investigated plasma levels of glutamine (the essential energy source of lymphcytes) to test the hypothesis that intensive training decreased plasma glutamine and thus NK cell cytotoxicity. METHODS: Eight college-level female volleyball players undertook one-month of heavy pre-season training 5h/day, 6 days per week. After an overnight fast, four morning resting blood samples were collected: pre-training, on the 10th day of training, one day before the end of training and one week after training, respectively. Plasma glutamine, glutamic acid and total amino acid concentrations were determined by HPLC. RESULTS: Plasma glutamine, glutamic acid and total aminoacid levels remained unchanged throughout the experiment. However, all glutamine concentrations (46.2–171.1 mmol/l) were substantially below the normal range (420–500 mmol/l). In contrast, glutamic acid levels were higher than normal, and total amino acid concentrations remained within the normal range. DISCUSSION: Our results do not explain training-induced changes in NK cell cytotoxicity, although low initial plasma glutamine levels may already have depressed NK cell cytotoxicity. CONCLUSIONS: One-month of heavy training did not change plasma glutamine levels of female volleyball players. These results do not support a link between plasma glutamine concentrations and decreased NK cell cytotoxicity during training. Supported by the Grant-in-Aid for Scientific Research C-2, 11680058, Japan Society for the Promotion of Science.

Enhancement of both cellular and humoral responses to genetic immunization by co-administration of an antigen-expressing plasmid and a plasmid encoding the pro-apoptotic protein Bax.

Transfecting cells with plasmid DNA encoding the protein Bax causes programmed cell death (apoptosis) and results in the formation of cell fragments (apoptotic bodies). It has been known for some time that when dendritic cells phagocytose apoptotic bodies derived from tumor cells, an immune response to tumor antigens can be generated. Gene expression in the skin was evaluated after intradermal injection with plasmids encoding fluorescent proteins. Plasmids encoding foreign antigens were co-injected intradermally with Bax-encoding plasmids or control plasmids to elicit both humoral and cytotoxic immunity. Immune responses to the antigens were assessed by ELISA and cytotoxicity assays. We demonstrate here that injection of a mixture of reporter gene plasmids into the skin results in the expression of both plasmids in the large majority of the transfected cells. It is known that immune responses to multiple antigens can be elicited by co-injection of separate individual plasmids. When mice were injected with equal quantities of two antigenic plasmids and either the Bax plasmid or a noncoding control plasmid, antibody responses were increased 4-8-fold in the Bax group. Similarly, cytotoxic T lymphocyte (CTL) responses in the Bax group showed an 80% increase in the number of lytic units per million cells. This data shows that simply including the apoptosis-inducing Bax plasmid along with antigen-expressing plasmids may provide a significant enhancement of immune responses to DNA vaccines. Published in 2004 by John Wiley & Sons, Ltd.