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Purpose: Patients with metastatic or recurrent Ewing’s sarcoma family of tumors and alveolar rhabdomyosarcoma have Experimental Design: Fifty-two patients with translocation positive, recurrent, or metastatic Ewing’s sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2.Results: All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2+ patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy.Conclusions: Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.

Supplementary Tables S1-S3 from A Pilot Study of Consolidative Immunotherapy in Patients with High-Risk Pediatric Sarcomas

[EN] Due to the heterogeneity of oils, the use of mixtures of lipases with di erent activity for a large number of glycerol-linked carboxylic acids that compose the substrate has been proposed as a better alternative than the use of one specific lipase preparation in the enzymatic synthesis of biodiesel. In this work, mixtures of lipases from di erent sources were evaluated in their soluble form in the ethanolysis of soybean oil. A mixture of lipases (50% of each lipase, in activity basis) from porcine pancreas (PPL) and Thermomyces lanuginosus lipase (TLL) gave the highest fatty acid ethyl ester (FAEE) yield (around 20 wt.%), while the individual lipases gave FAEE yields 100 and 5 times lower, respectively. These lipases were immobilized individually by the cross-linked enzyme aggregates (CLEAs) technique, yielding biocatalysts with 89 and 119% of expressed activity, respectively. A mixture of these CLEAs (also 50% of each lipase, in activity basis) gave 90.4 wt.% FAEE yield, while using separately CLEAs of PPL and TLL, the FAEE yields were 84.7 and 75.6 wt.%, respectively, under the same reaction conditions. The mixture of CLEAs could be reused (five cycles of 6 h) in the ethanolysis of soybean oil in a vortex flow-type reactor yielding an FAEE yield higher than 80% of that of the first batch., This work was supported by the São Paulo Research Foundation (FAPESP) (grant numbers #2011/23194-0 and #2016/10636-8), Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq, grant numbers #405889/2016-0, #308212/2017-7), in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001, and the MICIU of Spanish Government (project CTQ2017-86170-R).

Background: Vitamin D–binding protein (DBP) is the primary carrier of 25-hydroxyvitamin D [25(OH)D] in the circulation. One prospective study in male smokers found a protective association between DBP and pancreatic cancer, particularly among men with higher 25(OH)D concentrations. Objective: The objective was to examine the association between DBP and pancreatic cancer risk in an American population. Design: We conducted a nested case-control study in the Prostate, Lung, Colorectal, and Ovarian Cancer screening trial cohort of men and women aged 55–74 y at baseline. Between 1993 and 2010, 295 incident pancreatic adenocarcinoma cases were reported (follow-up to 15.1 y). Two controls (n = 590) were matched to each case by age, race, sex, and month of blood draw. We calculated smokingand diabetes-adjusted ORs and 95% CIs with the use of conditional logistic regression. Results: DBP concentration was not significantly associated with pancreatic cancer overall [highest ($7149.4 nmol/L) vs. lowest (,3670.4 nmol/L) quintile; OR: 1.75; 95% CI: 0.91, 3.37; P-trend = 0.25]. For serum 25(OH)D compared with the referent (50 to ,75 nmol/L), individuals in the highest group had a significantly higher risk ($100 nmol/L; OR: 3.23; 95% CI: 1.24, 8.44), whereas those in the lowest group had no significant association (,25 nmol/L; OR: 2.50; 95% CI: 0.92, 6.81). Further adjustment for DBP did not alter this association. Conclusion: Our results do not support the hypothesis that serum DBP or 25(OH)D plays a protective role in pancreatic cancer. This trial was registered at clinicaltrials.gov as NCT00339495. Am J Clin Nutr 2015;101:1206–15.

Here, we evaluated innate and adaptive immune system cytokine responses induced by HPV-16 L1 VLP in whole blood (WB) cultures from individuals receiving the vaccine (n=20) or placebo (n=4) before and after vaccination. 11 cytokines were measured: IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, and GM-CSF using multiplex bead arrays. Cytokine profiles from WB samples clearly discriminated between vaccine and placebo recipients and between pre and post-vaccination responses. Significant increases in Th1, Th2 and inflammatory cytokines were observed in WB assays following vaccination. Results from WB assays were compared against parallel PBMC-based assays in a subset of patients. Differences between whole blood assay and PBMC were observed, with the highest levels of induction found for WB for several cytokines. Our results indicate that multiplex assays for cytokine profiling in WB are an efficient tool for assessing broad spectrum, innate and adaptive immune responses to vaccines and identifying immunologic correlates of protection in efficacy studies.

Reduced breast cancer risk has been seen with the acquired immunodeficiency syndrome (AIDS) and rheumatoid arthritis.1–3 The CD4+ regulatory subset of T lymphocytes is a major focus of breast cancer pathogenesis,4, 5 but data on overall differences in peripheral blood mononuclear cell (PBMC) subsets between breast cancer cases and controls are sparse.6 We compared PBMC in 72 non-metastatic breast cancer cases (18 in situ, 54 locally invasive; mean age 54; all treated only with surgery) to 91 age-matched general-population controls in Warsaw and Lodz, Poland.7 Most cases were classified for tumor expression of estrogen receptor (ER), progesterone receptor (PR), HER2/neu (HER2), and epidermal growth factor receptor (EGFR). Each participant provided acid-citrate dextrose anticoagulated whole blood from which PBMC were frozen the same day in dimethyl sulfoxide and maintained at −80C until testing.8 PBMC immunophenotypes were determined by flow cytometry (Supplementary methods). Coefficients of variation (%CV) in duplicate vials were satisfactory (median 7.8%) except for the uncommon CD8+CD3- subset (36.7%). Case-control differences were tested by Kruskal-Wallis (KW) one-way analysis of variance and mixed models (Statistical Analysis System ver 9.0, Proc Mixed) that adjusted (adj) for 5-year age groups, city, and test date. Table 1 presents PBMC median values in the cases and controls. Total PBMC (98.5% lymphocytes) were modestly higher in cases than controls (14,132/µL vs 13,701/µL, PKW=0.04). CD19+ B cells were non-significantly higher, and CD3+ T cells were non-significantly lower among cases. Of the CD3+ T cells, cases tended to have lower levels of CD8+ cells (23.2% vs 26.5%, PKW=0.09), as well as non-significantly lower levels of CD4+ cells. CD4/CD8 ratio was non-significantly higher in cases (1.84) than controls (1.77, PKW=0.20). Table 1 Peripheral blood mononuclear cell (PBMC) phenotype levels in 72 breast cancer cases and 91 matched controls. Among the rarer T-cell populations, cases tended to have modestly higher levels of CD3+CD25+ cells (6.4% vs 5.3%, PKW=0.02), CD4+CD25+ cells (4.6% vs 4.2%, PKW=0.11), and CD4-FoxP3+ cells (0.20% vs 0.15%, PKW=0.05). Unexpectedly, CD4+FoxP3+ cells tended to be lower in cases (0.7% vs 1.1%, PKW=0.10). CD3+CD56+ natural killer (NK) T cells also tended to be lower in cases (5.8% vs 6.8%, P=0.12), in contrast to CD3-CD56+ NK cells, which were non-significantly higher in cases. Cases tended to have higher levels of CD80+ lymphocytes (2.1% vs 1.7%, PKW=0.06), as well as non-significantly higher levels of CD80+ monocytes (1.8% vs 1.2%, PKW=0.15). With multivariate adjustment for age, city, and test date, CD4/CD8 ratio was significantly higher in cases (Padj=0.001). With adjustment, cases and controls had no other significant differences, although cases tended to have more total PBMC (Padj=0.06) and fewer CD8+CD3+ T cells (Padj=0.06). Cases in all oncogene-defined strata (ER, PR, HER2, and EGFR) had more total PBMC than controls (PKW=0.01 to 0.06, Supplementary Table 1). Similarly, CD3+CD25+ cells were significantly elevated in all oncogene-defined strata of cases (PKW=0.01 to 0.05) except HER2-positive cases (PKW=0.56). CD4/CD8 ratio differed in all strata only after adjustment by mixed modeling; this was noteworthy with stratification for PR (Padj=0.0001). Other associations of nominal statistical significance (not presented) were either inconsistent across strata, or they were not confirmed by adjusted, mixed model analysis. Our population-based study found no major differences in PBMC levels between the general population of women with node-negative breast cancer and women randomly selected from the same general population. As observed elsewhere,6 CD8+ T cells tended to be lower in cases than in controls. Because cases had more total PBMC than did controls, a slightly higher deficit in total CD8+ cells among cases may have been hidden. Our study had several weaknesses, especially our use of frozen whole blood specimens. We used state-of-the-art flow cytometry that yielded highly reproducible results for common PBMC subsets in masked paired vials in the current study and previously,8 but accurate enumeration of rare cell populations may have been affected by freezing, thawing, and staining. We mitigated technical weaknesses by interspersing and identically handling specimens from cases and controls. Finally, we did not examine CXCR4, signaling of which may trigger apoptosis of neoplastic breast cells and account for the breast cancer deficit in women with AIDS.9, 10 This should be considered in future studies.

The potential role of vitamin D in cancer prevention has generated substantial interest, and laboratory experiments indicate several anti-cancer properties for vitamin D compounds. Prospective studies of circulating 25-hydroxyvitamin D [25(OH)D], the accepted biomarker of vitamin D status, suggest an inverse association with colorectal cancer risk, but with some inconsistencies. Furthermore, the direct or indirect impact of the key transport protein, vitamin D binding protein (DBP), has not been examined. We conducted a prospective study of serum 25(OH)D and DBP concentrations and colorectal cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, based on 476 colorectal cancer cases and 476 controls, matched on age, sex, race and date of serum collection. All subjects underwent sigmoidoscopic screening at baseline and once during follow-up. Conditional logistic regression estimated odds ratios (ORs) and 95% confidence intervals (CIs). Circulating 25(OH)D was inversely associated with colorectal cancer (OR = 0.60, 95% CI 0.38-0.94 for highest versus lowest quintile, p trend 0.01). Adjusting for recognized colorectal cancer risk factors and accounting for seasonal vitamin D variation did not alter the findings. Neither circulating DBP nor the 25(OH)D:DBP molar ratio, a proxy for free circulating 25(OH)D, was associated with risk (OR = 0.82, 95% CI 0.54-1.26, and OR = 0.79, 95% CI 0.52-1.21, respectively), and DBP did not modify the 25(OH)D association. The current study eliminated confounding by colorectal cancer screening behavior, and supports an association between higher vitamin D status and substantially lower colorectal cancer risk, but does not indicate a direct or modifying role for DBP.

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perforin and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7-AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of 51Cr release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly.

Background There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinicalscale method to prepare autologous DCs for cancer clinical trials. Methods PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1×10 6 cells/mL for 5–7 days at 37°C, with 5% CO 2 , with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heatinactivated allogeneicAB serumor autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. Results DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0±3.7 × 10 8 fresh or cryopreserved autologous monocytes, DC yield was 2.1±1.0 × 10 8 cells, or 29% of the starting monocyte number. Discussion In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1×10 6 cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

The Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, which is randomizing 74,000 screening arm participants (37,000 men, 37,000 women; ages 55-74) and an equal number of nonscreened controls, is a unique setting for the investigation of the etiology of cancer and other diseases and for the evaluation of potential molecular markers of early disease. At entry, baseline information is collected by questionnaire on dietary intake, tobacco and alcohol use, reproductive history (for women), family history of cancer, use of selected drugs, and other selected risk factors. Blood samples collected at the baseline screening exam are aliquoted to serum, plasma, red blood cell, and buffy coat fractions. At the next two annual screening visits, serum samples are collected. At the third annual reexamination, cryopreserved whole blood is obtained, in addition to serum, plasma, red blood cell, and buffy coat fractions. At the fourth and fifth years, serum, plasma, and buffy coat are collected. All blood samples are shipped to a central repository for long-term storage at -70 degrees C. Dietary questionnaires and buccal cells for DNA analysis are obtained from nonscreened controls. Cancer cases are identified through annual follow-up questionnaires, and all deaths are identified through vital status tracing mechanisms. Procedures are being developed to obtain archival pathologic material for selected cases of cancer and related diseases. Initial investigations are focusing on the etiology of colorectal cancer and on the operative characteristics of tests for the early detection of colorectal and prostate cancer.

We determined whether a classical conditioning paradigm may be used to condition immunologic responses in normal human subjects receiving an optimal immunostimulating dose of recombinant human interferon-gamma (rhIFN-gamma). We conducted a placebo-controlled, double-blind study of 31 normal volunteers in order to determine whether an initially immune-neutral stimulus, oral propylene glycol (PG), could eventually elicit an immune response as a consequence of its being paired with a known immunostimulatory dose and schedule of rhIFN-gamma. Subjects were randomly assigned to one of three groups: (A) rhIFN-gamma injections paired with PG; (B) normal saline injections paired with PG; (C) rhIFN-gamma injections alone. During the 4-week study, subjects received progressively fewer injections so that, by the final week of the study, no injections were given and groups A and B received only PG. The principal outcome measures were serum concentrations of quinolinic acid (QUIN) and neopterin, two nonspecific but sensitive markers of immune activation, and expression of Fc receptors (CD64) on peripheral blood mononuclear cells. RhIFN-gamma injections produced significant and predictable alterations in each of the measured immune parameters. No group B subject made an immune response. Mean serum QUIN levels were significantly higher at the end of week three for subjects in the experimental condition (group A) than for subjects receiving rhIFN-gamma alone (group C) despite receiving identical doses of rhIFN-gamma. Similarly, the predicted decay in mean serum neopterin levels from the end of week 1 to the end of week 2 was seen in group C but not in group A. The exposure of group A to PG blunted the decline of CD64 expression in week four. The data suggest that the pairing of an unconditioned stimulus (rhIFN-gamma) and a conditioned stimulus (PG) permits the conditioned stimulus alone to prolong a cytokine-induced response in normal humans.

The association of T-cell large granular lymphocyte (LGL) leukemia and rheumatoid arthritis is well described and it is now recognized that these patients and patients with Felty's syndrome represent different aspects of a single disease process. Most patients have rheumatoid arthritis at the time of diagnosis of LGL leukemia. This is the first detailed report of the development of rheumatoid arthritis after the diagnosis and treatment of LGL leukemia as well as the first report of rheumatoid arthritis that occurred in association with deoxycoformycin treatment. It is likely that the beneficial sustained normalization of neutrophil counts as a result of deoxycoformycin treatment played a significant role in the development of this complication. Hematological improvement occurred despite molecular genetic evidence of persistence of the abnormal T-cell clone. The role of the clonally expanded T cells in the pathogenesis of neutropenia and rheumatoid arthritis is discussed.

Benzene is known to have toxic effects on the blood and bone marrow, but its impact at levels below the U.S. occupational standard of 1 part per million (ppm) remains uncertain. In a study of 250 workers exposed to benzene, white blood cell and platelet counts were significantly lower than in 140 controls, even for exposure below 1 ppm in air. Progenitor cell colony formation significantly declined with increasing benzene exposure and was more sensitive to the effects of benzene than was the number of mature blood cells. Two genetic variants in key metabolizing enzymes, myeloperoxidase and NAD(P)H:quinone oxidoreductase, influenced susceptibility to benzene hematotoxicity. Thus, hematotoxicity from exposure to benzene occurred at air levels of 1 ppm or less and may be particularly evident among genetically susceptible subpopulations.

IgG-RFB4-SMPT-dgA consists of deglycosylated ricin A chain (dgA) coupled to the monoclonal antihuman CD22 antibody, RFB4. This study determined the maximally tolerated dose (MTD) of this immunotoxin (IT) administered as a continuous 8-day infusion to 18 patients with B-cell lymphoma (30% CD22+ tumor cells) over 8 days. The MTD was 19.2 mg/m2/192 h (maximum toxicity grade 1), with vascular leak syndrome (VLS) as dose-limiting toxicity (DLT) at 28.8 mg/m2/192 h (grades 3 through 5 in 7 of 11 patients). Predictors of severe VLS included serum IT concentrations greater than 1,000 ng/mL and the absence of circulating tumor cells. Decreased urine sodium excreted in 24 hours provided evidence for mild VLS without notable changes in serum albumin. Four partial responses, 3 minor responses, 6 stable disease, and 3 progression of disease were observed. The mean maximal serum concentration (Cmax) in initial courses at the MTD (19.2 mg/m2) was 443 +/- 144 ng/mL (n = 3; range, 326 to 604). At 28.8 mg/m2/192 h, the Cmax was highly variable (n = 11; mean, 1,102 +/- 702; range, 9.6 to 2,032 ng/mL). Human antimouse or antiricin antibodies developed in 6 of 16 (37.5%) patients after one course of IT. However, 10 eligible patients received multiple courses of IT. Changes in serum cytokines and cytokine receptors did not correlate with toxicity but decreased soluble interleukin-2 receptor concentrations correlated with clinical response. Comparison to a prior study with the same IT administered by intermittent bolus infusions (Amlot et al, Blood 82:2624, 1993) suggests similar clinical response, toxicity, and immunogenicity.

IL-15 promotes activation and maintenance of natural killer (NK) and CD8+ T effector memory (TEM) cells, making it a potential immunotherapeutic agent for the treatment of cancer and immunodeficiency states. Here we report the immunologic effects of 3 different IL-15 dosing strategies in Rhesus macaques. IL-15 at a dose of 20 μg/kg/d administered by continuous intravenous infusion for 10 days resulted in a massive (100-fold) expansion of CD8+ TEM cells in the peripheral blood. In contrast, the administration of 20-40 μg/kg/d of IL-15 by subcutaneous injection resulted in a more modest (10-fold) expansion of CD8+ TEM cells. NK expansion was similar in both the continuous intravenous and daily subcutaneous treatment groups. The observation that IL-15 administered by continuous intravenous infusion is able to induce markedly greater expansions of CD8+ TEM cells than the same dose administered by other routes may have important implications for clinical development of this cytokine.

We have defined a population of CD3-, CD56+ small lymphocytes (SLs) that exhibit the same phenotype and lytic capacity as natural killer (NK) cells. NK cells characteristically express the surface markers CD16 and CD56, mediate non–major histocompatibility complex (MHC)–restricted lysis, and have been equated with CD3- large granular lymphocytes (LGLs). In the present study we extended the observation that CD3-, CD56+ SLs can mediate NK- and antibody-dependent cellular cytotoxicity activity by studying the activation signals and lytic mechanisms that might be utilized by CD3-, CD56+ SLs in comparison to CD3- CD56+ LGLs. Our results show that CD3- SLs, similar to CD3- LGLs, exhibited activated killing in response to interleukin-2 (IL-2). In addition, after IL-2 activation, the CD3- SLs exhibited morphologic changes, including increases in size and granularity, and both morphologically and phenotypically became virtually indistinguishable from CD3- LGLs. Similar to CD3- LGLs, CD3- SLs could be directly activated by IL-2 alone to secrete significant quantities of interferon-γ (IFN-γ) and to express IL-2 receptor (IL-2R) p55. Examination of serine esterases and pore-forming protein (PFP) demonstrated that these cells exhibited a cytoplasmic distribution of perforin, which, unlike that of CD3- LGLs, was not associated with dense cytoplasmic azurophilic granules. Serine esterase levels were similar. However, after IL-2 activation PFP was concentrated in dense cytoplasmic granules, similar or identical to the situation in CD3-, CD56+ LGLs. These CD3-, CD56+ subsets appear to represent a continuum of activated cells that might represent various states of maturation of NK cells.

Objectives Deoxyribonucleic acid (DNA) extraction from blood and genotyping for candidate single nucleotide polymorphisms (SNP) is now an important part of almost all molecular epidemiologic studies. However, in many studies the amount of blood sample is limited or only serum is available. We conducted several pilot studies to identify methods for DNA extraction and high-throughput SNP genotyping of both white blood cell (WBC) and serum DNA that can be done centrally and reliably for large numbers of samples. Methods We used biospecimens from the Prostate Cancer Prevention Trial (PCPT), a phase III, double-blind, placebo-controlled trial that tested the efficacy of finasteride for the primary prevention of prostate cancer. DNA was extracted from WBCs, from serum, and also from serum after organic solvent extraction for analysis of hormones. We also conducted blinded high-throughput genotyping in three laboratories to assess feasibility and reliability of results with differing methodologies using DNA from WBCs and from serum. Results Genotyping of DNA extracted from WBCs resulted in highly reliable, reproducible results across laboratories using different genotyping platforms. However, genotyping with DNA extracted from serum did not provide reliable data using high-throughput multiplex approaches such as Sequenom (hME and iPLEX) and Applied Biosystems SNPlex, but was successful using Taqman. Conclusions Based on the results of these pilot studies, we conclude that DNA obtained from serum must be used judiciously, and that genotyping using multiplex methods is not suitable for serum DNA.

Leptin, the protein product of the ob gene, regulates appetite and body weight in animals. Endotoxin and cytokines, induced by endotoxin, interleukin (IL) 1 and tumor necrosis factor, increase expression of leptin in mice and hamsters. We measured serum leptin concentrations in patients with cancer before and after administration of recombinant human IL-1α. Fourteen patients received IL-1α at one of three dose levels (0.03, 0.1, or 0.3 μg/kg·day) for 5 days. Serum leptin concentrations increased in all but two patients within 24 h after the first dose. The increase in leptin was correlated directly with IL-1α dose (P = 0.0030). Despite continued administration of IL-1α, serum leptin concentrations returned to pretreatment levels by day 5 of therapy. An increase in serum leptin concentrations may be one mechanism by which anorexia is induced by IL-1α. However, tachyphylaxis of the leptin response suggests that other mechanisms also are involved.

Background With chronic infection, hepatitis C virus (HCV) RNA can be detected in B cells and associated with B-cell disorders, but these are not well defined. Methods The relationship between HCV infection and lymphocyte subpopulations was evaluated rigorously in 120 asymptomatic hemophilic patients, randomly selected from a prospective cohort study. CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells were quantified by flow cytometry using cryopreserved peripheral blood mononuclear cells of 24 hemophilic patients in each of five age-matched groups [uninfected; chronic HCV with or without human immunodeficiency virus (HIV); and cleared HCV with or without HIV]. Results As expected, patients with HIV had significantly reduced CD4+ and increased CD8+ T cells. Irrespective of HIV, patients with chronic HCV infection had approximately 25% fewer CD19+ B cells than those without chronic HCV infection. Conclusions These data support the hypothesis that asymptomatic patients with chronic HCV infection have an altered B-lymphocyte population.

The role of host immunity in the development of virus-induced cancers has been difficult to elucidate, in part because of our inability to effectively measure multiple immune parameters using available amounts of biological material. The objective of the present study was to validate the use of a newly developed multiplex assay, the LINCOplex assay, for the simultaneous measurement of multiple cytokines [interleukin/(IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-gamma, and tumor necrosis factor-alpha]. Supernatants obtained from peripheral blood mononuclear cell cultures stimulated with various different mitogens and antigens (including phytohemagglutinin, influenza, tetanus, HPV16 E6 and E7 peptides, and media alone) were selected for study. In total, 750 specimens obtained from 26 participants were tested in replicate using the 8-plex LINCOplex assay (25 micro l of specimen required per well). Every specimen was included in duplicate in a blinded fashion. For some specimens, multiple 2-fold dilutions of the same specimen were included to evaluate the linearity of results. The availability of independently obtained IL-2 and IFN-gamma results from standard single cytokine (simplex) assays allowed for a direct comparison between the LINCOplex results and those obtained from the simplex assays. Spearman correlation coefficients for continuous results, and exact agreement rates and weighted kappa statistics for quartiled variables, were computed to evaluate intra- and interassay agreement. IL-4 levels were consistently below the detectable level of the assay (3 pg/ml) whereas IL-6 and IL-8 levels were consistently above the highest detectable level of the assay (10,000 pg/ml), and these three cytokines were, therefore, not evaluated further. For the remaining five cytokines, excellent intra-assay reproducibility was observed, with Spearman correlation coefficients consistently above 0.90 for all five cytokines. Exact agreement rates ranging from 77.6-90.3% and weighted kappas ranging from 0.81-0.92 were observed. Similar results were observed when analysis was restricted to supernatants obtained from cultures that had been stimulated with HPV16 peptides and when analysis was restricted to supernatants obtained from cultures containing no antigen or mitogen, suggesting that the LINCOplex assay is applicable under conditions where moderate or weak cytokine responses/levels are expected. Linearity of results was observed when dilutions of a single specimen were blindly tested, with the exception of IL-2 and IL-10, where deviations from linearity were sometimes observed. For IL-2 and IFN-gamma, where results from simplex assays were available for comparison, the LINCOplex assay and the simplex assay results agreed well. Spearman correlation coefficients were 0.86 and 0.93 for IL-2 and IFN-gamma, respectively. Exact agreement and weighted kappa values were 68.5% and 0.72 (95% confidence interval, 0.65-0.79), respectively, for IL-2 and 67.3% and 0.73 (95% confidence interval, 0.67-0.80), respectively, for IFN-gamma. These results indicate the applicability of the LINCOplex assay for the simultaneous measurement of multiple cytokines using small amounts of biological material.

Clinical immunotherapy trials using DCs depend on large-scale methods for DC generation that fulfil current good manufacturing practice requirements. Our goal was to develop data on two variables, monocyte-enrichment method and culture container, which could be used to design a closed-system process for ex vivo generation of immature DCs.Mononuclear cells were collected by leukapheresis and enriched for monocytes by either counterflow centrifugal elutriation, or immunomagnetic selection using Isolex, an automated closed-system device. Monocytes were cultured for 7 days in serum-free medium with GM-CSF and IL-4, using either plastic flasks or gas-permeable Stericell bags. Monocytes and cultured DCs were evaluated for yield, flow cytometric phenotype, and in vitro function in MLR, and autologous recall responses to tetanus toxoid and influenza virus.Enriched monocyte products from elutriation and immunomagnetic selection were equivalent in yield and purity, and were capable of generating immature DCs in either flasks or bags. DCs from all four culture conditions were equivalent in yield, phenotype, and in vitro function. Mean DC yield was 67-80% per seeding monocyte, and 11-13% per starting mononuclear cell (MNC). A leukapheresis product containing 5 x 10(9) MNCs processed by this method could therefore yield approximately 5 x 10(8) immature DCs.In this manufacturing process, the Isolex system was equivalent to elutriation, and Stericell bags were equivalent to flasks. Together, the Isolex system and Stericell bags can be incorporated into a closed-system process to generate immature DCs.

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.

Dendritic cells (DCs) are potent antigen presenting cells reported to undergo irreversible functional 'maturation' in response to inflammatory signals such as TNF-alpha. The current paradigm holds that this DC maturation event is required for full functional capacity and represents terminal differentiation of this cell type, culminating in apoptotic cell death. This provides a possible mechanism for avoiding dysregulated immunostimulatory activity, but imposes constraints on the capacity of DCs to influence subsequent immune responses and to participate in immunological memory. We report that the cell surface and functional effects induced by TNF-alpha are reversible and reinducible. These effects are accompanied by a concordant modulation of cytokine mRNA expression that includes the induction of proinflammatory factors (IL-15, IL-12, LT-alpha, LT-beta, TNF-alpha, RANTES) which is coincident with the down-regulation of counter-regulatory cytokines (IL-10, TGF-beta1, TGF-beta2, IL-1 RA, MCP-1). The resultant net effect is a dendritic cell activation state characterized by a transient proinflammatory posture. These results demonstrate that 1) human DCs do not undergo terminal 'maturation' in response to TNF-alpha, 2) DC phenotypes are more pleiotropic than previously thought, and 3) DCs are potential immunoregulatory effector cells with implications for control of immune responses in both in vivo and in vitro systems.

Research examining the association between vitamin D and pancreatic cancer is inconsistent, with some studies showing an elevated risk with increased vitamin D (Stolzenberg-Solomon, 2006, 2009, 2010) and another showing an inverse association. (Wolpin, 2012) This counterintuitive finding has led researchers to investigate levels of vitamin D-binding protein (DBP) as a possible explanation. DBP serves several important functions in the body, including acting as the primary carrier of vitamin D in the bloodstream. (Speeckaert, 2006) Previous research in the Alpha-Tocopherol, Beta-Carotene Study found an inverse trend for DBP and pancreatic cancer in male Finnish smokers, with higher compared to lower DBP concentrations showing a protective association with pancreatic cancer. (Weinstein, 2012) We conducted a nested case-control study in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial cohort of men and women aged 55-74 at baseline. Between 1993 and 2010, 295 incident cases of pancreatic adenocarcinoma were reported (follow-up to 15.1 years). Two controls (n = 590) who were alive at the time the case was diagnosed were selected for each case and were matched by age, race, sex, month of blood draw. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using conditional logistic regression, adjusting for smoking status and diabetes mellitus. DBP concentration was not significantly associated with pancreatic cancer overall (highest vs. lowest quintile, ≥7149.4 vs.5 years after baseline (n = 214 cases, highest vs. lowest quintile, OR=2.12, 95% CI 1.01-4.43, p-value=0.047). We did not confirm the protective association seen previously. Our results do not support the hypothesis that DBP plays a protective role in pancreatic cancer. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Marina R. Piper, William Kopp, Helen Rager, Ronald L. Horst, Rachael Z. Stolzenberg-Solomon. Vitamin D-binding protein and pancreatic cancer: A nested case-control study. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B61.

The adoptive transfer of anti-CD3-stimulated T killer (T-AK) cells was tested with different bolus and infusional interleukin-2 (IL-2) regimens, and anti-CD3 stimulation procedures to determine immunologic and antitumor effects in patients with a variety of advanced cancers. Indium-111 labeling was used to observe traffic patterns of the infused T-AK. Autologous peripheral blood mononuclear cells were obtained by leukapheresis. Cyclophosphamide (300 mg/m2) was given to most patients immediately after leukapheresis. The harvested cells were activated ex vivo with anti-CD3 overnight or for 4 days, at which time cells were reinfused and an IL-2 regimen was begun. Treatment was repeated 28 days later. This treatment regimen induced significant increases in leukocytes, lymphocytes, and eosinophils in patients in most treatment cohorts. Circulating lymphocytes were predominantly CD3+ T cells with preferential expansion of the CD8+ subset. Patients receiving cells stimulated in vitro for 4 days had significant T-cell lymphocytosis with either infusional or bolus plus infusional IL-2 regimens. T-cell viability was decreased in culture after a second 4-day stimulation with anti-CD3 at day 28; this decrease could be prevented by adding IL-2 to the culture media. Cells stimulated overnight required both bolus and infusional IL-2 to show an atypical lymphocytosis in vivo. Overnight-stimulated T-AK did not show decreases in in vitro viability at the day 28 restimulation. Indium-III-labeled cells trafficked to the liver, spleen, and bone marrow. No increase in uptake was observed in tumor deposits. There were 2 patients with partial responses, 5 with minor responses, 19 with stable disease, and 88 with progressive disease. The length of in vitro anti-CD3 stimulation, and the dose and timing of IL-2 administration in vivo results in different circulating leukocyte populations after adoptive T-AK infusion. Generally, the CD8+ T-cell subset was preferentially expanded by this treatment approach. Repeated ex vivo stimulation with anti-CD3 may cause cell death.

Hypertensive factor (HF), a compound isolated from the erythrocytes of rats and tentatively identified as a peptide, has been shown to influence tissue calcium metabolism and induce prolonged blood pressure elevation. In the present study, we investigated the biological properties of antibodies directed against this peptide. Partially purified antibody preparations significantly decreased HF stimulation of lanthanum-resistant calcium uptake in rat aortic tissue in vitro. Infusion of the antibody preparation into spontaneously hypertensive (SH) or normotensive Sprague–Dawley rats resulted in a rapid decline in mean blood pressure of 54 and 34 Torr (1 Torr = 133.332 Pa), respectively. In contrast, infusion of the serum immunoglobulin preparations from controls (unimmunized and ovalbumin-immunized rabbits) had no significant effect on the blood pressure of SH or normotensive rats. The systolic blood pressure of SH rats was reduced for at least 72 h following a single injection of the antibody preparations, whereas the blood pressure of normotensive rats had returned to normal levels within 24 h following antibody injection. The results indicate that the anti-HF antibody preparation antagonizes the stimulation of calcium uptake by the peptide and acutely lowers blood pressure in SH and normotensive rats.Key words: antibodies, blood pressure regulation, hypertensive factor, spontaneously hypertensive rat.