Dendritic Cell-Based Cancer Vaccines.

Dendritic cells (DC) are specialized immune cells that play a critical role in promoting an immune response against Ags, which can include foreign pathogenic Ags and self-tumor Ags. DC are capable of boosting a memory T cell response but most importantly they are effective initiators of naive T cell responses. Many years of studies have focused on the use of DC vaccines against cancer to initiate and shape an antitumor-specific immune response and/or boost existing spontaneous antitumor T cell responses. In this study we give a brief overview of DC biology, function, and cellular subsets, and review the current status of the field of DC as cancer vaccines.

Climate risk and seasonal forage production of Marandu palisadegrass in Brazil.

This study aimed to characterize Brachiaria brizantha cv. Marandu seasonal production (seasonality) and its variation (climate risk) yearlong throughout Brazil. Data from weather stations in Brazil (1963-2009), were associated with an empirical herbage accumulation rate (HAR; kg DM ha-1 day-1) model which considers growing degree-days adjusted by a drought attenuation index. Simulations were performed under 20, 40, 60 and 100 mm of soil water holding capacities (SWHCs). HAR's means and standard deviations were calculated for the seasons of the year. Thereafter, cluster analysis and calculations were performed to gather similar weather stations and characterize seasonality and climate risk indexes. Cluster analysis resulted in four Groups per SWHC. The north of Brazil (Group 1) presented the lowest seasonality and climate risk indexes and low need for precautions. In the middle west (Group 2), the seasonality index ranged from medium-high to high. Winter and Summer presented the lowest and highest production, respectively. In the south of Brazil, some regions in the southeast and northeast (Group 3), Winter presented the lowest production and highest climate risk index, probably due to low temperatures. The northeast (Group 4) presented a seasonality index that ranged from medium-high to very high and low productions.

Hepatocellular cancer-derived alpha fetoprotein uptake reduces CD1 molecules on monocyte-derived dendritic cells.

Alpha fetoprotein (AFP) is produced by over 50% of hepatocellular carcinomas (HCC). Uptake of tumor-derived AFP (tAFP) can impair activity of human dendritic cells (DC). The expression pattern of the lipid antigen presenting genes from the CD1 family is reduced in AFP-treated monocyte-derived DC. Surface CD1 family proteins, particularly CD1d, were reduced in AFP-exposed DC (by both normal cord blood-derived AFP (nAFP) and tAFP). NKT cells recognize lipid antigens presented by CD1d molecules. They play an important role in connecting the innate and adaptive immune systems, and in anti-tumor immunity. We hypothesized that AFP might impair the ability of DC to stimulate natural killer T (NKT) cells. No significant impact of AFP was observed on NKT cell stimulation. By examining secreted cytokines, we observed non-significant AFP-induced changes in several secreted proteins. These data indicate that AFP downregulates CD1 molecules on DC, but the impact on NKT cell activations is minimal.

Cell-intrinsic in vivo requirement for the E47-p21 pathway in long-term hematopoietic stem cells.

Major regulators of long-term hematopoietic stem cell (LT-HSC) self-renewal and proliferation have been identified, but knowledge of their in vivo interaction in a linear pathway is lacking. In this study, we show a direct genetic link between the transcription factor E47 and the major cell cycle regulator p21 in controlling LT-HSC integrity in vivo under repopulation stress. Numerous studies have shown that E47 activates p21 transcription in hematopoietic subsets in vitro, and we now reveal the in vivo relevance of the E47-p21 pathway by reducing the gene dose of each factor individually (E47(het) or p21(het)) versus in tandem (E47(het)p21(het)). E47(het)p21(het) LT-HSCs and downstream short-term hematopoietic stem cells exhibit hyperproliferation and preferential susceptibility to mitotoxin compared to wild-type or single haploinsufficient controls. In serial adoptive transfers that rigorously challenge self-renewal, E47(het)p21(het) LT-HSCs dramatically and progressively decline, indicating the importance of cell-intrinsic E47-p21 in preserving LT-HSCs under stress. Transient numeric recovery of downstream short-term hematopoietic stem cells enabled the production of functionally competent myeloid but not lymphoid cells, as common lymphoid progenitors were decreased, and peripheral lymphocytes were virtually ablated. Thus, we demonstrate a developmental compartment-specific and lineage-specific requirement for the E47-p21 pathway in maintaining LT-HSCs, B cells, and T cells under hematopoietic repopulation stress in vivo.

Getting pastoral systems productivity right.

Beef production in pasture-based systems is increasingly contested due to related biophysical and environmental challenges. Addressing these requires rigorous science-based evidence to inform private decisions and public policies. Increasing yields and simultaneously reducing the negative environmental impacts of agricultural and livestock production are central to sustainable intensification approaches. Yet, stocking rate, the commonly used metric for animal productivity in pastures, or more broadly, of sustainable intensification in pastoral production systems, warrants scrutiny to signpost successful transformative change of food systems and to avoid provision of misleading policy advice. Here we discuss why future studies would benefit of considering the two constituent elements of productivity in pastoral systems - animal performance (kg of animal product/head) and stocking rates (heads/ha) -, rather than stocking rates alone.

Comparative Analysis of 5-Year Clinical Outcomes and Patterns of Failure of Proton Beam Therapy Versus Intensity Modulated Radiation therapy for Prostate Cancer in the Postoperative Setting.

PURPOSE: Although proton beam therapy (PBT) is a rapidly expanding modality to treat prostate cancer compared with intensity modulated radiation therapy (IMRT), data comparing disease control outcomes and patterns of failure in the postprostatectomy setting remain substantially limited. METHODS AND MATERIALS: All patients who underwent postoperative IMRT or PBT to the prostate bed only at a single institution were included (2009-2017). Endpoints included biochemical failure (BF; using institutional and recent cooperative group trial definitions), local failure (LF), regional failure (RF), distant failure (DF), and all-cause mortality. A case-matched cohort analysis was performed using 3-to-1 nearest-neighbor matching; multivariable Cox proportional hazards modeling (MVA) estimated hazard ratios for disease-related outcomes by treatment modality. RESULTS: Of 295 men, 260 were matched (n = 65 PBT, 195 IMRT); after matching, only age at diagnosis (P < .01) significantly differed between cohorts. At a median follow-up of 59 months, BF (institution-defined), LF, RF, DF, and mortality rates were 45% (n = 29), 2% (n = 1), 9% (n = 6), 9% (n = 6), and 2% (n = 1) for PBT, and 41% (n = 80), 3% (n = 5), 7% (n = 13), 9% (n = 18), and 5% (n = 9) for IMRT (all P > .05). RT modality was not significantly associated with BF on MVA using institutional or cooperative group definitions (all P > .05), nor with LF (P = .82), RF (P = .11), DF (P = .36), or all-cause mortality (P = .69). Patterns of failure were qualitatively similar between cohorts (DF: bone, retroperitoneal nodes, lung). CONCLUSIONS: In this single institution, case-matched analysis, PBT yielded similar long-term disease-related outcomes and patterns of failure to IMRT in the postprostatectomy setting.

Investigating immune and non-immune cell interactions in head and neck tumors by single-cell RNA sequencing.

Head and neck squamous cell carcinoma (HNSCC) is characterized by complex relations between stromal, epithelial, and immune cells within the tumor microenvironment (TME). To enable the development of more efficacious therapies, we aim to study the heterogeneity, signatures of unique cell populations, and cell-cell interactions of non-immune and immune cell populations in 6 human papillomavirus (HPV)+ and 12 HPV- HNSCC patient tumor and matched peripheral blood specimens using single-cell RNA sequencing. Using this dataset of 134,606 cells, we show cell type-specific signatures associated with inflammation and HPV status, describe the negative prognostic value of fibroblasts with elastic differentiation specifically in the HPV+ TME, predict therapeutically targetable checkpoint receptor-ligand interactions, and show that tumor-associated macrophages are dominant contributors of PD-L1 and other immune checkpoint ligands in the TME. We present a comprehensive single-cell view of cell-intrinsic mechanisms and cell-cell communication shaping the HNSCC microenvironment.

Inhibition of mitochondrial respiratory chain in the brain of adult rats after acute and chronic administration of methylphenidate.

Methylphenidate (MPH) is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. It was previously demonstrated that MPH altered brain metabolic activity. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. However, there are still few studies about MPH effects on the brain of adult rats. Thus, in the present study we evaluated the effect of acute or chronic administration of MPH on the activities of mitochondrial respiratory chain complexes I-IV in the brain of adult rats. For acute administration, a single injection of MPH was given to 60-day-old rats. For chronic administration, MPH injections were given to 60-day-old rats once daily for 28 days. Our results showed that complexes I, II, III and IV were inhibited after acute or chronic MPH administration in the hippocampus, prefrontal cortex, striatum and cerebral cortex. On the other hand, cerebellum was not affected.

Evaluation of brain creatine kinase activity in an animal model of mania induced by ouabain.

Bipolar disorder (BD) is a common and severe mood disorder associated with higher rates of suicide and disability. The development of new animal models, and the investigation employing those available have extensively contributed to understand the pathophysiological mechanisms of BD. Intracerebroventricular (i.c.v.) administration of ouabain, a specific Na+,K+-ATPase inhibitor, has been used as an animal model for BD. It has been demonstrated that Na+,K+-ATPase is altered in psychiatric disorders, especially BD. Creatine kinase (CK) is important for brain energy homeostasis by exerting several integrated functions. In the present study,we evaluated CK activity in the striatum, prefrontal cortex and hippocampus of rats subjected to i.c.v. administration of ouabain. Adult male Wistar rats received a single i.c.v. administration of ouabain (10(-2) and 10(-3) M) or vehicle (control group). Locomotor activity was measured using the open field test. CK activity was measured in the brain of rats immediately (1 h) and 7 days after ouabain administration. Our results showed that spontaneous locomotion was increased 1 h after ouabain administration and that hyperlocomotion was also observed 7 days after that.Moreover, CK activity was inhibited immediately after the administration of ouabain in the striatum, hippocampus and prefrontal cortex. Moreover, the enzyme was not affected in the striatum and hippocampus 7 days after ouabain administration. On the other hand, an inhibition in CK activity in the prefrontal cortex was observed. If inhibition of CK also occurs in BD patients, it will be tempting to speculate that the reduction of brain metabolism may be related probably to the pathophysiology of this disease.

Effect of therapeutic pulsed ultrasound on lipoperoxidation and fibrogenesis in an animal model of wound healing.

Evidence from the literature has shown that the wound healing process is enhanced by ultrasound therapy. In the present study, we measured thiobarbituric acid-reactive substances (TBARS; index of lipoperoxidation) and hydroxyproline (index of collagen synthesis) levels in wounds after therapeutic pulsed ultrasound (TPU) treatment. Male Wistar rats were submitted to skin ulceration, and three doses of TPU (0.4, 0.6, and 0.8W/cm(2)) were used. A circular area of skin was removed with a punch biopsy from the medial dorsal region. After TPU for 10 days, TBARS (Draper and Hadley [21]) and hydroxyproline (Woessner [22]) levels were measured in the tissue around the wound. Results showed that TPU improved wound healing, since the wound size was significantly smaller 5 and 10 days after ulceration in groups submitted to this treatment. Moreover, TBARS levels were decreased in the 0.4, 0.6, and 0.8W/cm(2) TPU groups, and hydroxyproline levels were increased in the 0.6 and 0.8W/cm(2) TPU groups. These findings indicate that TPU presents beneficial effects on the wound healing process, probably by speeding up the inflammatory phase and inducing collagen synthesis.

Dysregulated NF-κB-Dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Patients with Melanoma.

Therapeutic cancer vaccines targeting melanoma-associated antigens are commonly immunogenic but are rarely effective in promoting objective clinical responses. To identify critical molecules for activation of effective antitumor immunity, we have profiled autologous dendritic cell (DC) vaccines used to treat 35 patients with melanoma. We showed that checkpoint molecules induced by ex vivo maturation correlated with in vivo DC vaccine activity. Melanoma patient DCs had reduced expression of cell surface inducible T-cell costimulator ligand (ICOSL) and had defective intrinsic NF-κB signaling. Chromatin immunoprecipitation assays revealed NF-κB-dependent transcriptional regulation of ICOSL expression by DCs. Blockade of ICOSL on DCs reduced priming of antigen-specific CD8+ and CD4+ T cells from naïve donors in vitro Concentration of extracellular/soluble ICOSL released from vaccine DCs positively correlated with patient clinical outcomes, which we showed to be partially regulated by ADAM10/17 sheddase activity. These data point to the critical role of canonical NF-κB signaling, the regulation of matrix metalloproteinases, and DC-derived ICOSL in the specific priming of cognate T-cell responses in the cancer setting. This study supports the implementation of targeted strategies to augment these pathways for improved immunotherapeutic outcomes in patients with cancer.

Impact of checkpoint blockade on cancer vaccine-activated CD8+ T cell responses.

Immune and molecular profiling of CD8 T cells of patients receiving DC vaccines expressing three full-length melanoma antigens (MAs) was performed. Antigen expression levels in DCs had no significant impact on T cell or clinical responses. Patients who received checkpoint blockade before DC vaccination had higher baseline MA-specific CD8 T cell responses but no evidence for improved functional responses to the vaccine. Patients who showed the best clinical responses had low PD-1 expression on MA-specific T cells before and after DC vaccination; however, blockade of PD-1 during antigen presentation by DC had minimal functional impact on PD-1high MA-specific T cells. Gene and protein expression analyses in lymphocytes and tumor samples identified critical immunoregulatory pathways, including CTLA-4 and PD-1. High immune checkpoint gene expression networks correlated with inferior clinical outcomes. Soluble serum PD-L2 showed suggestive positive association with improved outcome. These findings show that checkpoint molecular pathways are critical for vaccine outcomes and suggest specific sequencing of vaccine combinations.

Investigating association of perineural invasion on prostate biopsy with Gleason score upgrading at prostatectomy: A multi-institutional analysis.

BACKGROUND: The significance of perineural invasion (PNI) in prostate cancer (PC) is unclear. A recent report of patients with pT2N0R0 PC found that PNI at prostatectomy was independently associated with higher Gleason score and more diffuse prostatic disease. We aimed to test our hypothesis that PNI on prostate biopsy in pT2N0R0 patients is associated with increased Gleason score upgrading at prostatectomy. METHODS: We identified 2892 patients status post prostatectomy with pT2N0R0 PC from three institutions, diagnosed between 1 January 2008 and 31 December 2014. Multivariable logistic regression (MVA) was used to evaluate the association between prostate biopsy PNI status and surgical Gleason upgrading, while controlling for potential confounders. RESULTS: Of the 2892 patients identified, 14% had PNI on biopsy, of whom 21% had surgical Gleason upgrading, while 28% without PNI on biopsy had such upgrading (P < .01). On MVA, the odds ratio (OR) of surgical Gleason upgrading for patients with biopsy PNI relative to patients without biopsy PNI was 0.69 (P < .01). The variables associated with surgical Gleason upgrading were age ≤60 years (OR 1.22, P = .02) and preoperative PSA >4 ng/mL (OR 1.26, P = .02). CONCLUSIONS: In post-prostatectomy patients with favorable-risk PC, PNI on prostate biopsy was not associated with surgical Gleason score upgrading. This may be due to the association of PNI with more diffuse disease, leading to increased biopsy tumor yield and grading accuracy. These findings suggest that in this setting, biopsy PNI alone should not be a concern for more aggressive disease requiring pathologic confirmation or intervention. This may help guide treatment decision-making for men debating active surveillance, radiation, and surgery.

Tumor-Derived α-Fetoprotein Suppresses Fatty Acid Metabolism and Oxidative Phosphorylation in Dendritic Cells.

Cellular metabolism supports immune cell function. Here, we identify a reduction in fatty acid synthesis and mitochondrial metabolism in dendritic cells (DC) due to α-fetoprotein (AFP), a protein secreted by hepatocellular cancer (HCC). DCs cultured in the presence of AFP show reduced expression of the metabolic regulatory molecules SREBP-1 and PGC1-α. The negative effect of AFP on mitochondrial metabolism and ATP production was confirmed with observation of reduction in basal oxygen consumption rate (OCR) in DCs exposed to AFP derived from cord blood. More severe reduction in basal OCR was observed in tumor-derived DCs exposed to AFP due to downregulation of cytochrome c oxidase. We also showed reduced expression of PGC1-α in circulating myeloid DCs of patients with HCC and impaired capacity to stimulate antigen-specific effector functions. These data show the negative effects of AFP on DC metabolism. These findings elucidate a mechanism of immune suppression in HCC and may help generate therapeutic approaches to reverse such immunosuppression.

CD56dim CD16- Natural Killer Cell Profiling in Melanoma Patients Receiving a Cancer Vaccine and Interferon-α.

Natural killer (NK) cells are innate cytotoxic and immunoregulatory lymphocytes that have a central role in anti-tumor immunity and play a critical role in mediating cellular immunity in advanced cancer immunotherapies, such as dendritic cell (DC) vaccines. Our group recently tested a novel recombinant adenovirus-transduced autologous DC-based vaccine that simultaneously induces T cell responses against three melanoma-associated antigens for advanced melanoma patients. Here, we examine the impact of this vaccine as well as the subsequent systemic delivery of high-dose interferon-α2b (HDI) on the circulatory NK cell profile in melanoma patients. At baseline, patient NK cells, particularly those isolated from high-risk patients with no measurable disease, showed altered distribution of CD56dim CD16+ and CD56dim CD16- NK cell subsets, as well as elevated serum levels of immune suppressive MICA, TN5E/CD73 and tactile/CD96, and perforin. Surprisingly, patient NK cells displayed a higher level of activation than those from healthy donors as measured by elevated CD69, NKp44 and CCR7 levels, and enhanced K562 killing. Elevated cytolytic ability strongly correlated with increased representation of CD56dim CD16+ NK cells and amplified CD69 expression on CD56dim CD16+ NK cells. While intradermal DC immunizations did not significantly impact circulatory NK cell activation and distribution profiles, subsequent HDI injections enhanced CD56bright CD16- NK cell numbers when compared to patients that did not receive HDI. Phenotypic analysis of tumor-infiltrating NK cells showed that CD56dim CD16- NK cells are the dominant subset in melanoma tumors. NanoString transcriptomic analysis of melanomas resected at baseline indicated that there was a trend of increased CD56dim NK cell gene signature expression in patients with better clinical response. These data indicate that melanoma patient blood NK cells display elevated activation levels, that intra-dermal DC immunizations did not effectively promote systemic NK cell responses, that systemic HDI administration can modulate NK cell subset distributions and suggest that CD56dim CD16- NK cells are a unique non-cytolytic subset in melanoma patients that may associate with better patient outcome.

Multiple antigen-engineered DC vaccines with or without IFNα to promote antitumor immunity in melanoma.

BACKGROUND: Cancer vaccines are designed to promote systemic antitumor immunity and tumor eradication. Cancer vaccination may be more efficacious in combination with additional interventions that may build on or amplify their effects. METHODS: Based on our previous clinical and in vitro studies, we designed an antigen-engineered DC vaccine trial to promote a polyclonal CD8+ and CD4+ T cell response against three shared melanoma antigens. The 35 vaccine recipients were then randomized to receive one month of high-dose IFNα or observation. RESULTS: The resulting clinical outcomes were 2 partial responses, 8 stable disease and 14 progressive disease among patients with measurable disease using RECIST 1.1, and, of 11 surgically treated patients with no evidence of disease (NED), 4 remain NED at a median follow-up of 3 years. The majority of vaccinated patients showed an increase in vaccine antigen-specific CD8+ and CD4+ T cell responses. The addition of IFNα did not appear to improve immune or clinical responses in this trial. Examination of the DC vaccine profiles showed that IL-12p70 secretion did not correlate with immune or clinical responses. In depth immune biomarker studies support the importance of circulating Treg and MDSC for development of antigen-specific T cell responses, and of circulating CD8+ and CD4+ T cell subsets in clinical responses. CONCLUSIONS: DC vaccines are a safe and reliable platform for promoting antitumor immunity. This combination with one month of high dose IFNα did not improve outcomes. Immune biomarker analysis in the blood identified several predictive and prognostic biomarkers for further analysis, including MDSC. TRIAL REGISTRATION: NCT01622933 .

Training Intraverbal Naming to Establish Matching-to-Sample Performances.

The current study evaluated whether training intraverbal naming would be sufficient to establish visual-visual matching-to-sample (MTS) performances in college students. In the first experiment, we used a multiple-probe design across stimulus sets to assess whether six participants could match arbitrary visual stimuli (AB) after learning to tact their two experimentally defined classes (A' and B') and then intraverbally relate their names (i.e., "A' goes with B'"). All participants matched the stimuli accurately after training, as well as emitted the trained intraverbals. In the second experiment, we used a multiple baseline design across four participants to assess whether the same training would produce bi-directional intraverbals in the form of "B' goes with A'," and MTS performance consistent with symmetry (BA). All participants responded accurately during matching and intraverbal tests. Across both experiments, participants stated the trained intraverbals while performing the matching task. Results showed that MTS performance can be established solely by verbal behavior training.

Molecular resolution of the B cell landscape.

The progression of hematopoietic stem cells (HSCs) to the B lymphocyte lineage requires that uncommitted progenitors successfully negotiate the transition from multipotency to unipotency, including the loss of self-renewal potential. Previous work identified essential transcription factors that mediate B lineage development. Major advances build on this knowledge and reveal coordinated changes in gene expression occurring within single cells at sequential stages in the B cell differentiation pathway. Recent studies on epigenetic mechanisms also provide a framework within which transcription factor activity, chromatin modifications, and gene expression patterns can be viewed at hierarchical levels to link genotype and phenotype.

Evaluation of Krebs cycle enzymes in the brain of rats after chronic administration of antidepressants.

Several works report brain impairment of metabolism as a mechanism underlying depression. Citrate synthase and succinate dehydrogenase are enzymes localized within cells in the mitochondrial matrix and are important steps of Krebs cycle. In addition, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase and succinate dehydrogenase activities from rat brain after chronic administration of paroxetine, nortriptiline and venlafaxine. Adult male Wistar rats received daily injections of paroxetine (10mg/kg), nortriptiline (15mg/kg), venlafaxine (10mg/kg) or saline in 1.0mL/kg volume for 15 days. Twelve hours after the last administration, the rats were killed by decapitation, the hippocampus, striatum and prefrontal cortex were immediately removed, and activities of citrate synthase and succinate dehydrogenase were measured. We verified that chronic administration of paroxetine increased citrate synthase activity in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected. Chronic administration of nortriptiline and venlafaxine did not affect the enzyme activity in these brain areas. Succinate dehydrogenase activity was increased by chronic administration of paroxetine and nortriptiline in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected either. Chronic administration of venlafaxine increased succinate dehydrogenase activity in prefrontal cortex, but did not affect the enzyme activity in cerebellum, hippocampus, striatum and cerebral cortex. Considering that metabolism impairment is probably involved in the pathophysiology of depressive disorders, an increase in these enzymes by antidepressants may be an important mechanism of action of these drugs.