Lytic Units Research Articles

Evaluation of the DNA topoisomerase inhibitor irinotecan (CPT-11) as an alternative to irradiation for pretreatment of NK-92s prior to adoptive cell immunotherapy.

Abstract Irradiation damages both proteins and DNA, which reduces the cytotoxicity of NK-92 cells as well as preventing their growth and engraftment when used for adoptive immunotherapies of cancer. We evaluated the DNA topoisomerase inhibitor irinotecan (CPT-11) as an alternative method to 10 Gy irradiation that might better preserve the perforin-granzyme cytotoxicity mediated by NK-92 cells towards tumor cells. Irinotecan is enzymatically converted into an active metabolite SN-38 which promotes its potential to become a delayed inhibitor in comparison to camptothecin which is a structurally similar topoisomerase inhibitor that is immediately active. We pretreated NK-92 cells for 4 hours with different concentrations of CPT-11, and measured NK-92 cell death and the effects on cell-mediated cytotoxicity towards Raji tumor cells (measured by 51Cr release one day after pretreatment). Previous experiments with 10 Gy irradiation were used for comparative purposes. Irinotecan had a dose response on cell death which progressed for 5 days. We found that one day after pretreatment with as little as 80 – 100 uM CPT-11, viable cell recoveries were similar to 10 Gy irradiated with over 50% compared to the counts of control cells. Activity of the viable cells after CPT-11 pretreatment, compared by lytic units, was over 30% conserved whereas irradiated cell activity was 2.9%, compared to controls. This data demonstrate that the negative effects of anti-proliferative treatment are less pronounced with irinotecan, than for irradiation. We conclude that optimization of dosage and exposure times to irinotecan may offer a means to prolong the life and function of NK-92 cells that are used for adoptive immunotherapies. Supported by the Nevada INBRE NIH GM103440, for student summer research scholarship and the UNR Foundation C.E. Hudig Fund.

Clinical and laboratory signs of haemophagocytic lymphohistiocytosis associated with pandemic influenza A (H1N1) infection in patients needing extracorporeal membrane oxygenation: A retrospective observational study.

Severe pandemic influenza has been associated with the hyperinflammatory condition secondary haemophagocytic lymphohistiocytosis (HLH). To determine the frequency, degree, character and possible cause of influenza-associated HLH in critically ill patients with severe acute respiratory distress syndrome due to influenza A (H1N1) infection requiring extracorporeal membrane oxygenation (ECMO) support at our hospital. A retrospective observational study. Medical data were retrieved retrospectively from 11 consenting patients of thirteen adults infected with pandemic influenza A (H1N1) 2009 requiring ECMO between July 2009 and January 2010 at the ECMO Centre of Karolinska University Hospital, Stockholm, Sweden. All patients were evaluated for HLH using HLH-2004 criteria and HScore. Eleven patients (median age 31 years) were included in the study and all survived. All patients showed signs of multiple organ dysfunction and pronounced inflammation, more severe in the four patients with HLH who had significantly higher peak serum concentrations of ferritin (P = 0.024), alkaline phosphatase (P = 0.012) and gamma-glutamyl transferase (P = 0.024), lower concentration of albumin (P = 0.0086) and more frequently hepatomegaly (P = 0.048). Abnormal lymphocyte cytotoxicity (lytic units <10) and a low proportion of natural killer (NK) cells were observed in three of four patients with HLH. Notably, we found a significant inverse correlation between serum ferritin concentration and NK cell and cytotoxic T lymphocyte percentages (rs = -0.74, P = 0.0013 and rs = -0.79, P = 0.0025, respectively). One HLH patient received HLH-directed cytotoxic therapy, another intravenous immunoglobulin and the other two no specific HLH-directed therapy. Critically ill patients, including healthy young adults, with pandemic influenza may develop HLH and should be monitored for signs of hyperinflammation and increasing organ dysfunction, and evaluated promptly for HLH because HLH-directed therapy may then be beneficial. The association of low NK percentages with hyperferritinaemia may suggest a role for reduced NK cell numbers, possibly also cytotoxic T lymphocytes, and subsequently reduced lymphocyte cytotoxicity, in the pathogenesis of hyperinflammation and secondary HLH.

Laparoscopic Surgery Versus Open Surgery for Colorectal Cancer: Impacts on Natural Killer Cells.

Background: Laparoscopic resection is increasingly used in colorectal cancer (CRC). It has been suggested to carry short-term benefits in safety, recovery, and preservation on immune function for patients with CRC. However, the impact of laparoscopic resection on natural killer (NK) cells is largely unclear. Methods: A total of 200 patients with CRC across Dukes A/B/C stages were randomly assigned to laparoscopic or open resection. The blood samples were collected before and after the surgery. The total number of NK cells was quantified by flow cytometer. Lytic units 35 toward K562 was used to quantify NK cells activity. The outcomes between the groups across pathological stages were also analyzed. Results: The number and activity of NK cells decreased after the surgery in both groups. The laparoscopic group showed a faster recovery rate of NK cells function than the control group as assessed by cell count and lytic activity. Natural killer cells were impaired in a higher degree in patients at Dukes B/C stages. The recovery of NK cells to baseline level at day 7 postsurgery was observed in the laparoscopic group across all 3 stages. Conclusion: Generally, laparoscopically assisted surgery resulted in a better preservation on NK cells function. A better outcome was observed in patients with CRC at Dukes B/C stages.

Effects of short-term Pilates exercise on selected blood parameters

The aim of our prospective, interventional, pre-post, single arm study was to supplement the lack of knowledge of the effect of short-term Pilates intervention on selected blood parameters of healthy women. Female volunteers were recruited for 2-weeks Pilates intervention. Blood has been collected and anthropometric parameters were measured before and after exercise period (EP). Plasma insulin, cortisol, and dehydroepiandrosterone sulphate levels, erythrocyte antioxidant activity, glutathione levels, NK cytotoxicity and plasma cytokines were analysed. We found a decrease in erythrocyte antioxidant enzymes SOD and GPx activity; GSH levels; in the pro-inflammatory chemokine MCP-1 and trend to reduction in MIP-1β, PDGF and VEGF levels in plasma. NK cell cytotoxic activity increased after Pilates EP in the percentage of specific lysis at 25:1 effector: target (E:T) ratio and the same trend was observed at all E:T ratios as well as in the amount of lytic units per 107 cells. Our findings show that Pilates exercise may improve NK cell immune response and inflammatory milieu in plasma of healthy women.

An improved method to quantify human NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) per IgG FcR-positive NK cell without purification of NK cells

Natural killer (NK) lymphocyte ADCC supports anti-viral protection and monoclonal antibody (mAb) anti-tumor therapies. To predict in vivo ADCC therapeutic responses of different individuals, measurement of both ADCC cellular lytic capacity and their NK cellular receptor recognition of antibodies on ‘target’ cells are needed, using clinically available amounts of blood. Twenty ml of blood provides sufficient peripheral blood mononuclear cells (PBMCs) for the new assay for lytic capacity described here and for an antibody EC50 assay for Fc-receptor recognition. For the lytic capacity assay, we employed flow cytometry to quantify the CD16A IgG Fc-receptor positive NK effector cells from PBMCs to avoid loss of NKs during isolation. Targets were 51Cr-labeled Daudi B cells pretreated with excess obinutuzumab type 2 anti-CD20 mAb and washed; remaining free mAb was insufficient to convert B cells in the PBMCs into ‘targets’. We calculated: the percentage Daudis killed at a 1:1 ratio of CD16A-positive NK cells to Daudis (CX1:1); lytic slopes; and ADCC50 lytic units. Among 27 donors, we detected wide ranges in CX1:1 (16–73% targets killed) and in lytic slopes. Slope variations prevented application of lytic units. We recommend CX1:1 to compare individuals' ADCC capacity. CX1:1 was similar for purified NK cells vs. PBMCs and independent of CD16A V & F genotypes and antibody EC50s. With high mAb bound onto targets and the high affinity of obinutuzumab Fc for CD16A, CX1:1 measurements discern ADCC lytic capacity rather than antibody recognition. This assay allows ADCC to be quantified without NK cell isolation and avoids distortion associated with lytic units.

56 - A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity

Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. When the target cells are lysed by the immune cells (effector), they release the entrapped radioactive 51Chromium, which is measured to determine the level cytotoxicity induced by the effector cells. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First, live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was used to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined by normalizing results based on time = 0. The donor PBMCs were added to each well with Calcein-stained target cells at different Effector-to-Target (E:T) ratios. The 96-well microplate was then scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. Although it could be argued that release of a dye may not actually represent cytotoxicity by effector cells, nonetheless much like 51Cr release assay percent cytotoxicity was directly proportionate to the duration of the assay, E:T ratio used, and amount of activating cytokine present in the media, all as expected. Further, when PBMCs from both donors showed near equal level of cytotoxicity at 4 hr; upon calculation of the NK cell lytic unit, it was revealed that one donor possessed much higher level of effector function than the other . The proposed image cytometry method can scan and analyze the entire well area of the entire 96-well plate in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.

Quantitative assay of human antibody-dependent cell-mediated cytotoxicity (ADCC) using peripheral blood mononuclear cells (PBMCs)

Abstract ADCC can be essential for anti-viral immunity and supports many tumor immunotherapies. It is a challenge to quantify and one wants a fast assay that uses few lymphocytes when patient blood is limited. Three considerations affected our new assay design: 1) normal B cells within PBMCs introduce ‘cold target’ competition of radiolabeled tumor B cells if anti-B cell antibodies are present in the ADCC assays; 2) type 1 antibodies to CD20 B cell epitopes are internalized and cleared from the cell surface while type 2 anti-CD20 antibodies remain external; and 3) target cells with MHC I initiate KIR-regulation of lysis that will vary among donors. To address these issues, we used Daudi B leukemia cells that lack MHC I and pretreated the Daudi cells with the humanized nonfucosylated/glycoengineered monoclonal type 2 anti-CD20 antibody obinutuzumab (GA101 G2). Target cell pretreatment prevented ‘cold target’ competition. We used 4 hr 51Cr-release for its sensitivity and varied the PBMC to target (E:T) cell ratios to obtain lytic units. Flow cytometric counts of CD16A Fc-receptor positive cells with TrueCountR beads determined the exact number of ADCC effectors within the unfractionated PBMCs. There was low NK activity to Daudi cells, which required subtraction of simultaneous controls without antibody. We plotted ADCC activity as a log function of the E:T ratios and observed that ADCC was dependent upon interactions of multiple CD16A-positive effectors per target cell, which was expressed as lytic units per 106 CD16A cells, that varied 50 fold among donors. In addition, the linear slopes of CD16A cell lytic cooperativity varied 8 fold. In summary, we have established a fast quantitative assay for comparison of human ADCC that uses as few as 8 million PBMCs.

Abstract 428: Dual E-selectin and CXCR4 inhibition reduces tumor growth and increases the sensitivity to docetaxel in experimental bone metastases of prostate cancer

Abstract Prostate cancers preferentially metastasize to the skeleton where the bone microenvironment can stimulate excessive tumor cell growth and spread, and promote the emergence of clinically-resistant disease. An improved understanding of the complex relationship between prostate carcinoma (PCa) cells and the bone microenvironment has created a powerful opportunity to develop novel therapies. PCa cells preferentially roll and adhere on bone marrow vascular endothelial cells, where constitutive E-selectin expression and abundant stromal cell-derived factor-1α (SDF-1α) are expressed and interact with E-selectin ligands and CXCR4 present on PCa cells. These molecular interactions initiate a cascade of activation events that lead to the development of treatment resistant metastases. This suggests that agents able to antagonize these molecular interactions may be used as pharmacological treatments of bone metastatic disease. In the current studies we investigate if a dual E-selectin/CXCR4 inhibitor (GMI-1359) could impact the intraosseous growth of the metastatic, androgen-independent PC3M cell line and affect chemosensitivity to docetaxel. PCa cells, including PC3M, selected for increased visceral and bone metastatic potential express high levels of E-selectin ligands and CXCR4 as compared to nonmetastatic PCa cell lines. We evaluated the ability of GMI-1359 administered alone or in combination with docetaxel to inhibit the growth and metastasis of intratibially implanted luciferase-transfected PC-3M cells. Approximately two weeks post tumor cell implantation, mice were treated by intraperitoneal injection for 2 weeks with either saline twice daily; 40 mg/kg GMI-1359 twice daily, 5 mg/kg docetaxel once weekly or a combination of GMI-1359 and docetaxel. Thirty-five days after initiation of treatment, the percentage of tibiae positive by X-ray and the size of osteolytic lesions was impacted by treatment with GMI-1359 alone or in combination with docetaxel. Docetaxel alone had only a modest impact on intraosseous lesions. Lytic units were reduced by 38%, 78% and 88% in mice treated with docetaxel alone, GMI-1359 alone, or GMI-1359 in combination with docetaxel, respectively. The significantly reduced intraosseous growth of PC3M cells correlated with decreased serum levels of both mTRAP and type I collagen fragments. Our data provides a clear biologic rationale for the use of a dual E-selectin/CXCR4 inhibitor as an adjuvant to taxane-based chemotherapy in men with high-risk prostate cancer to prevent bone metastases. Given its complementary mechanism of action to traditional chemotherapy, GMI-1359 warrants further development not only in prostate carcinoma, but also in other malignancies where tumor cells are likely to spread to bone. Citation Format: Giovanni L. Gravina, Andrea Mancini, Alessandro Colapietro, Simona D. Monache, Adriano Angelucci, Alessia Calgani, William E. Fogler, John L. Magnani, Claudio Festuccia. Dual E-selectin and CXCR4 inhibition reduces tumor growth and increases the sensitivity to docetaxel in experimental bone metastases of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 428. doi:10.1158/1538-7445.AM2015-428

Immune markers predictive of neuropsychiatric symptoms in HIV-infected youth.

The purpose of this study was to evaluate possible associations between systemic immune dysregulation (activated CD8(+) T lymphocytes and natural killer [NK] cell count/function) and symptoms of depression and anxiety in youth with horizontally (behaviorally) acquired HIV infection. This secondary analysis of a previously collected prospective cohort included 323 youth with horizontally acquired HIV infection enrolled in the Reaching for Excellence in Adolescent Care and Health (REACH) cohort of the NICHD/NIH. A multivariable linear regression model with generalized estimating equations for intraindividual repeated measures was used to examine the relationship between flow cytometry measurements of activated T lymphocytes (CD8(+) CD38(+)), NK cells (CD3(-) CD16(+) CD56(+)), and NK cell functional activity (lytic units per NK cell and per peripheral blood mononuclear cell) and their association with subsequent symptoms of depression (Center for Epidemiologic Studies depression scale) and anxiety (Revised Children's Manifest Anxiety Scale). Higher measures of NK cell functional activity were associated with fewer anxiety symptoms measured 12 months later in crude and adjusted analyses. Higher counts of activated T cells were associated with fewer depression symptoms measured 12 months later in adjusted analysis. NK cell function and activated T-lymphocyte count may be related to subsequent symptoms of depression and anxiety.

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

Cell-based flow cytometry assay to measure cytotoxic activity.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

Comparison of Antibodies That Mediate HIV Type 1 gp120 Antibody-Dependent Cell-Mediated Cytotoxicity in Asymptomatic HIV Type 1-Positive Men and Women

Recent studies suggest that HIV-specific antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies contribute to protective immunity against HIV. An important characteristic of future HIV vaccines will, therefore, be the ability to stimulate production of these antibodies in both men and women. Early studies suggest that men may have a better ADCC antibody response against HIV than women. Our objective was to determine whether men and women differ with respect to their ADCC response to HIV-1 gp120. HIV-positive, asymptomatic untreated men and women were matched for race, age, CD4(+) T cell number, HIV-1 viral load, and treatment and HIV-1 gp120 ADCC antibody titers were compared. A standard (51)Cr-release assay was used to determine HIV-1 gp120 ADCC antibody titers in HIV-1-seropositive individuals from the Multicenter AIDS Cohort Study (MACS; n=32) and the Women's Interagency HIV Study (WIHS; n=32). Both sexes had high ADCC titers against HIV-1 gp120: 34.4% (n=11) and 40.6% (n=13) of men and women, respectively, had titers of 10,000; 62.5% (n=20) and 56.3% (n=18) had titers of 100,000. Groups did not differ in percent specific release (% SR), lytic units (LU), correlations of titer to viral load, or titer to CD4(+) T cells in men or women. Both groups also had similar cross-clade ADCC antibody responses (p>0.5 for % SR and LU). Comparable groups of asymptomatic HIV-1-infected men and women had comparable HIV-1 gp120 ADCC antibodies. Both sexes had significant cross-clade reactivity. Differences between men and women may become evident as disease progresses; this should be evaluated at later stages of HIV-1 infection.

Abstract 1252: Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia.

Abstract Introduction: In the treatment of acute leukemia, much of the therapeutic effect of allogeneic stem cell transplantation is centered on engraftment of donor cells into the host, with a resultant graft versus leukemia effect. Work previously done by our group and others has shown the infusion of haploidentical stem cells in patients with acute leukemia results in a cytokine storm and exhibits an anti-tumor effect in the absence of engraftment. Whether the underlying clinical response was due to this cytokine storm or secondary to host or donor effector cell interactions was not determined. We present in vitro data collected from leukemia patients in which patient CD3+ cells are stimulated with randomly selected unrelated normal donor peripheral blood mononuclear cells (PBMC). The stimulated patient CD3+ cells are then collected and tested for their ability to lyse leukemia cells from the patient. Methods: New leukemia patients were identified through standard pathologic diagnostic criteria. Patient samples were collected and separated into CD3+ and CD3- fractions. Patient CD3+ cells were then placed in a mixed lymphocyte culture and stimulated with mitomycin C treated unrelated normal donor PBMC. Cell proliferation was measured on Day 5. On Day 7, the stimulated patient CD3+ cells were collected and then cultured with Chromium 51 labeled CD3- cells, the bulk of which were leukemic blast cells. Cytolytic activity was measured through Cr51 release assays. Cell lysis was recorded as lytic units/million cells (LU/106), which is inversely related to the effector to target ratio at which 30% cell lysis occurs. Patient demographic data including sex and age in addition to leukemia type, cytogenetics, and molecular markers were recorded. Results: 10 total patients were enrolled in the study; 8 AML, 1 CML, and 1 CMML. 9 patients underwent ex vivo CD3+ stimulation with 2 separate donor PBMC samples while 1 sample underwent only 1 donor stimulation for 19 total assays. The average white blood cell count was 57,640 cells/uL (range 7,100-336,500 cells/ul) with 4-80% peripheral blasts. 7 out of 19 (37%) assays showed leukemia cell cytolytic activity greater than 1 lytic unit. All 7 successful assays were derived from patients with AML (Table 1). Conclusions: Patient CD3+ cells stimulated with random unrelated donor PBMC are able to generate a cytolytic anti-leukemia response in a donor dependant fashion. This cytolytic activity appears to be unrelated to host lymphocyte proliferation upon donor antigenic stimulation. Interestingly, of the seven assays with cytolytic activity greater than one lytic unit, four were performed in the two patients with known FLT3 ITD positive leukemia, which has a notoriously poor prognosis. Further studies to elucidate a mechanism of action for cellular therapy are warranted. Citation Format: John L. Reagan, Loren D. Fast, Martha Nevola, Peter J. Quesenberry. Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1252. doi:10.1158/1538-7445.AM2013-1252

Rational Design of Engineered Cationic Antimicrobial Peptides Consisting Exclusively of Arginine and Tryptophan, and Their Activity against Multidrug-Resistant Pathogens

The emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against both P. aeruginosa and S. aureus under challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens.

A Randomized Placebo-Controlled Trial of Massage Therapy on the Immune System of Preterm Infants

The aim of this study was to investigate the effects of massage therapy (MT) on the immune system of preterm infants. The primary hypothesis was that MT compared with sham therapy (control) will enhance the immune system of stable premature infants by increasing the proportion of their natural killer (NK) cell numbers. A randomized placebo-controlled trial of MT versus sham therapy (control) was conducted among stable premature infants in the NICU. Study intervention was provided 5 days per week until hospital discharge for a maximum of 4 weeks. Immunologic evaluations (absolute NK cells, T and B cells, T cell subsets, and NK cytotoxicity), weight, number of infections, and length of hospital stay were also evaluated. The study enrolled 120 infants (58 massage; 62 control). At the end of the study, absolute NK cells were not different between the 2 groups; however, NK cytotoxicity was higher in the massage group, particularly among those who received ≥5 consecutive days of study intervention compared with control (13.79 vs 10 lytic units, respectively; P = .04). Infants in the massage group were heavier at end of study and had greater daily weight gain compared with those in the control group; other immunologic parameters, number of infections, and length of stay were not different between the 2 groups. In this study, MT administered to stable preterm infants was associated with higher NK cytotoxicity and more daily weight gain. MT may improve the overall outcome of these infants. Larger studies are needed.

Abstract 3515: Maitake D-Fraction, a natural mushroom extract, synergizes with Interleukin-2 for increased lytic activity of peripheral blood mononuclear cells against various human tumor cell histologies

Abstract Maitake (Grifola frondosa) has been recognized by naturopathic physicians as a medicinal mushroom capable of immune support. Maitake D-Fraction (MDF), a component of the maitake mushroom isolated during hot-water extraction, is rich in α-Glucans and commonly prescribed as a dietary supplement. Because MDF is widely available and easily tolerated, it is highly desirable for the cancer patient looking for immune support via natural products. Previous research has suggested that a dietary MDF regimen can enhance immune function in humans. However, studies to investigate the capacity of MDF to increase tumor immunity are lacking. This study was designed to investigate whether human peripheral blood mononuclear cells (PBMC) treated with MDF develop increased cytolytic capacity against human tumor cells. Cytolysis mediated by MDF-activated PBMC was compared to that of naïve PBMC (media control) and PBMC that were treated with interleukin-2 (IL-2). Immune mediated tumor cytolysis was assessed used a standard Chromium-Release Assay and expressed as number of lytic units (LU)/10^7 effector cells. Target cells for the assays included the PANC1 pancreatic cancer cell line (PA) purchased from ATCC, and single cell suspensions created from peritoneal carcinomatosis (PC) and renal cell carcinoma (RC) tumors resected from patients. Against the PA cells, the LU observed were 16.9 (media control), 12.9 (1ug/ml MDF), 23.8 (10ug/ml MDF) and 28.9 (100ug/ml MDF). The corresponding LU values against PC cells were 7.6, –13.4, –14.2, and –12.1, respectively, and against RC cells 12.3, 2.7, 2.0, and 8.2, respectively. Predictably, PBMC stimulated with an optimally activating dose of IL2 (200U/ml) exhibited a marked increase in LU against all target cells. Nevertheless, when PBMC were stimulated concurrently with both IL-2 and MDF activation was increased further. Thus, LU against PA target cells were 73.0 for PBMC treated with IL-2 alone, and 110.6 for PBMC treated with IL-2 plus 100ug/ml MDF. The corresponding LU values against PC target cells were 37.1 (IL-2 alone), and 66.4 (IL-2 plus 100ug/ml MDF) and against RC target were 94.0 (IL-2 alone) and 110.7 (IL-2 plus 100ug/ml MDF). This study shows that MDF has the capacity to increase the lytic activity produced by an optimally activating dose of IL-2, eliciting greater killing of a variety of human cancer cells than what can be achieved with IL2 alone. It is reasonable to assume that MDF can elicit a similar effect at lower doses of IL-2. Given the toxicity of IL-2 observed in humans, the potential to elicit the desired immune stimulation by combining lower doses of IL-2 with MDF becomes highly desirable. Future clinical trials should be designed to investigate the ability of MDF to elicit optimal immune stimulation with more tolerable doses and schedules of IL-2. 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 3515. doi:1538-7445.AM2012-3515

Abstract 1552: Resveratrol increases sensitivity of human peritoneal cancer cells to macrophage-mediated cytolysis

Abstract The peritoneal cavity contains numerous cells of the immune system capable of mediating antitumor effects. The persistence of cancer in this environment is thought to be due, in part, to the capacity of these cells to circumvent immunologic detection and destruction. Nevertheless, the presence of an immune system within the peritoneal cavity provides the opportunity to elicit tumor cytolysis by resident and/or circulating immune cells mobilized from the peripheral blood. One strategy that may be applicable in this setting is the introduction of compounds into the peritoneal cavity that enhance the sensitivity of malignant cells to activated immune cells. Ideally, these compounds would be tolerated by normal tissues while exerting clinical effects on tumor tissues. In the current investigation, we explored the capacity of the natural product, resveratrol, to modulate sensitivity of human peritoneal cancer cells to the lytic effects of activated human peripheral blood monocytes (PBM). PBM were primed with gamma interferon (IFN) followed by lipopolysaccharide (LPS) as a second signal. Target cells consisted of newly established cells lines from surgical specimens of patients with peritoneal carcinaomatosis (PC) or metastatic colorectal cancer (CRC). Cytolysis was determined following activation with a 24 hour 51Chromium release assay. Control activation of PBM with medium elicted 25 lytic units (LU) and &amp;lt; 5 LU against untreated CRC and PC cells respectively. These values were not changed significantly when resveratrol-pretreated tumor cells were used as targets. In contrast, PBM fully activated with IFN/LPS elicted 224 and 118 LU against untreated CRC and PC respectively and 316 and 199 LU against resveratrol-pretreated CRC and PC respectively. These results were investigated further to determine the effects of resveratrol pretreatment on expression of genes associated with immunologic sensitivity. A real time PCR assay indicated significantly increased expression of death receptors of the TNF superfamily as a consequence of resveratrol pretreatment. Subsequent studies demonstrated that recombinant TNF and TRAIL ligands could significantly inhibit proliferation of resveratrol-pretreated CRC and PC greater than media-pretreated cells. The results of this study demonstrate that human peritoneal cancer cells can be rendered significantly more sensitive to the cytolytic effects of activated human monocytes and macrophages when exposed to the natural product, resveratrol. Given the substantial quantity of macrophages in the peritoneal cavitry of humans, and the influence of malignant tissues on increasing these numbers further, modalities designed to exploit the tumor cytolytic effects of peritoneal macrophages for the treatment of human peritoneal cancers should be explored. 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 1552. doi:1538-7445.AM2012-1552

Daily intake ofLactobacillus caseiShirota increases natural killer cell activity in smokers

Dietary probiotics supplementation exerts beneficial health effects. Since cigarette smoking reduces natural killer (NK) activity, we evaluated the effect of Lactobacillus casei Shirota (LcS) intake on NK cytotoxic activity in male smokers. The double-blind, placebo-controlled, randomised study was conducted on seventy-two healthy Italian blue-collar male smokers randomly divided for daily intake of LcS powder or placebo. Before and after 3 weeks of intake, peripheral blood mononuclear cells were isolated and NK activity and CD16⁺ cells' number were assessed. Daily LcS intake for 3 weeks significantly increased NK activity (P < 0.001). The increase in NK activity was paralleled by an increase in CD16⁺ cells (P < 0.001). Before intake, NK cytotoxic activity inversely correlated with the number of cigarettes smoked (R - 0.064). LcS intake prevented the smoke-dependent expected NK activity reduction. The analysis of the distribution of changes in smoke-adjusted NK activity demonstrated that the positive variations were significantly associated with LcS intake, while the negative variations were associated with placebo intake (median value of distributions of differences, 20.98 lytic unit (LU)/10⁷ cells for LcS v. - 4.38 LU/10⁷ cells for placebo, P = 0.039). In conclusion, 3 weeks of daily LcS intake in Italian male smokers was associated with a higher increase in cytotoxic activity and CD16⁺ cells' number in comparison to the placebo intake group.

Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells

The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activity is frequently measured using percentage of specific lysis calculations that do not quantify actual target cell death and effector numbers at the single cell level. Here we established a new flow cytometry-based assay that allows accurate single-cell analysis of cytotoxic capacity of primary antigen-specific CD8 T cells generated in vivo in humans after antigenic exposure without in vitro amplification that can be used for specificities restricted by different HLAs as target cells are autologous cells. We show that this assay is robust, highly sensitive irrespective of the frequency of antigen-specific CD8 T cells, and allows accurate calculation of the index of cytotoxic capacity in lytic units. This new assay provides a sensitive method to measure the intrinsic cytotoxic activity of antigen-specific CD8 T cells directly ex vivo on human primary cells.

Abstract 1809: Stimulation of antibody dependent cell-mediated cytotoxicity by interleukin-15 is equivalent to interleukin-2

Abstract An important strategy for the treatment of cancer involves administration of tumor-specific antibodies that target tumor cells for destruction by the individual's leukocytes via antibody dependent cell-mediated cytotoxicity (ADCC). However, ADCC is frequently depressed in individuals with cancer. Interleukin-2 (IL-2), a pro-inflammatory cytokine, enhances ADCC and is used clinically for this purpose. Interleukin-15 (IL-15) is a cytokine that shares many functions with IL-2, including stimulation of ADCC and proliferation and activation of natural killer cells. In contrast to IL-2, IL-15 supports the survival of CD8+ memory T cells, does not produce T cell activation-induced cell death and has no marked effects on Treg cells. Since these different functions may prove beneficial when using a cytokine to stimulate ADCC in vivo in cancer immunotherapy, we compared the in vitro effect of IL-15 (generously provided by Dr. Stephen Creekmore, NCI-Frederick) versus IL-2 to enhance ADCC. Using a standard 51Cr release assay, M21 human melanoma cells were loaded with 51Cr and incubated for four hours at 37° with human peripheral blood mononuclear cells (PBMC), hu14.18K322A, (generously provided by Dr. Fariba Navid of St. Jude Children's Research Hospital) a monoclonal antibody specific for GD2 (disialoganglioside) that is overexpressed on M21 cells (5 ng/ml), and either media, IL-2 (10 ng/ml) or IL-15 (10 ng/ml). The ratio of effectors (PBMCs) to targets (M21) was 10:1 or 30:1 and % cytotoxicity was determined from which lytic units were calculated. Incubation with IL-2 augmented ADCC from 163±39 to 230±40 lytic units (n=6, mean±SEM) while incubation with IL-15 enhanced ADCC from 148±27 to 229±36 lytic units (n=6). ADCC response was comparable for IL-2 and IL-15 (p=0.4857) and similar results were obtained using higher and lower concentrations of hu14.18K322A (0.1 – 100 ng/ml) and IL-2 and IL-15 (5 – 25 ng/ml), and after pre-incubation of PBMCs with IL-2 or IL-15 (4 – 44 hours). These experiments indicate that IL-2 and IL-15 demonstrate comparable stimulation of ADCC in vitro. If IL-15 produces equivalent enhancement of ADCC compared to IL-2 in vivo, it may be a potential immunotherapeutic for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1809. doi:10.1158/1538-7445.AM2011-1809