CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

Factors associated with adolescent HPV vaccination in the U.S.: A systematic review of reviews and multilevel framework to inform intervention development.

Multilevel factors impact HPV vaccine series initiation and completion among adolescents in the U.S. Synthesis of these factors is needed to inform intervention development and to direct future research. Current frameworks synthesizing factors focus on females only and do not include both series initiation and completion outcomes. We conducted a systematic review of reviews to identify modifiable individual-, provider-, and clinic-level factors associated with HPV vaccination outcomes among U.S. adolescents and developed a multilevel framework illustrating relations between factors to inform intervention development. We searched Medline, PsychInfo, Pubmed, CINAHL, and ERIC databases and included reviews published 2006 to July 2, 2018 describing individual-, provider-, or clinic-level factors quantitatively associated with HPV vaccination among U.S. adolescents. Two coders independently screened reviews, extracted data, and determined quality ratings. Sixteen reviews containing 481 unique primary studies met criteria. Factors synthesized into the multilevel framework included parent psychosocial factors (knowledge, beliefs, outcome expectations, intentions) and behaviors, provider recommendation, and patient-targeted and provider-targeted clinic systems. The scope of our framework and review advances research in two key ways. First, the framework illustrates salient modifiable factors at multiple levels on which to intervene to increase HPV vaccination. Second, the review identified critical gaps in the literature at each level. Future research should link the body of literature on parental intentions to vaccination outcomes, identify provider psychosocial factors associated with recommendation behaviors and subsequent vaccine uptake in their patient population, and understand clinic factors associated with successful implementation of patient- and provider-targeted system-level interventions.

Orbital Reconstruction in Neuroectodermal Tumor of the Orbit: Multimodal Treatment Approach.

INTRODUCTION: Primitive neuroectodermal tumors of peripheral origin are very rare, and orbital neuroectodermal tumors are even more uncommon. Only 25 patients with primary orbital involvement in the pediatric age group have been reported. METHODS: In this article, the authors describe their experience in the multimodality treatment approach to treat neuroectodermal tumor of the orbit. The authors also present a male patient 3-year old presenting with a neuroectodermal tumor of the right orbit causing rapidly progressive proptosis. The patient underwent an upper and lateral orbital marginotomy. The entire bone defect was reconstructed with a bone graft, allowing for the reconstruction of the floor and the lateral wall of the middle cranial fossa, the floor of the anterior cranial fossa, the upper and lateral orbital frame, and the right zygomatic bone. Over a period of 16 months, the patient was subjected to chemotherapy. RESULTS: In the postoperative period, a favorable evolution of the disease was observed, with growth in the reconstructed structures, good projection of the orbit and the eyeball, and stable results without tumor recurrence. CONCLUSIONS: The authors present the clinical analysis, surgical management, as well as the chemotherapy treatment established, with follow-ups at 1 and 2 and a half years. This experience shows the effectiveness of multimodality therapy in the treatment of rare tumors of difficult handling.

Ammonia and greenhouse gases losses from mechanically turned cattle manure windrows: A regional composting network.

An on-farm composting network operates in the Basque Country (northern Spain), in which solid manure produced in livestock farms (mostly dairy and beef cattle) is composted through windrow turning. This network aims to produce a valuable resource (compost) for the farmers whereas the volume of the solid manure was reduced at farm level The objective of the study was to assess the gaseous losses (NH3 and GHG) from 6 on-farm composting windrows (either deep litter systems or solid fraction after slurry separation) after turning operations. Monitored turning events occurred 1 to 4 months after establishing the heaps on the field. Ammonia and greenhouse gas (GHG) losses were estimated by the open and close chamber techniques, respectively. Results showed overall low emission rates related to the long degradation period of the windrows. Maximum NH3 release was at 2.0 mg m-2 d-1 after the second/third turning events. Baseline N2O losses were below 50 mg m-2 d-1, with maximum rates close to 500 mg m-2 d-1 some days after turning works. Methane emissions were mostly below 100 mg m-2 d-1, while CO2 losses were lower than 25 g m-2 d-1. Carbon dioxide peaks (≈250 g m-2 d-1) were reached after the second/third turnings. Overall, gaseous N and C losses accounted for 0.1 and 1% of the initial N and C content of the windrows, respectively. The present study concluded that two/three turning operations in aged solid manure-derived compost windrows do not have significant effects on NH3 and GHG losses. The magnitude of the gaseous losses from on-farm composting systems is dependent on the manure management practices at farm level (e.g. moment of windrow stacking).

In-Silico Analyses of Molecular Force Sensors for Mechanical Characterization of Biological Systems.

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for in situ force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide- and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Förster resonance energy transfer (FRET), linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Cα atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.

Functional testing, coronary artery calcifications, and outcomes in Hodgkin lymphoma survivors treated with chest radiation.

BACKGROUND: Consensus guidelines recommend periodic screening for coronary artery disease (CAD) in Hodgkin lymphoma (HL) survivors treated with radiation therapy (RT) to the chest. However, the prognostic utility of screening strategies in this population remains unclear. We evaluated the association between functional testing, coronary artery calcifications (CAC), and guideline-based risk assessment and major adverse cardiovascular events (MACE) in HL survivors treated with RT. METHODS: We retrospectively studied HL survivors treated with RT who underwent functional testing between 2003 and 2020 and chest computed tomography (CT) within 12 months of each other at our center. CAC was assessed semi-quantitatively from CT images. Cardiovascular risk was estimated using the 2019 ACC/AHA Guideline on the Primary Prevention of Cardiovascular Disease. Diagnostic test characteristics were calculated using major adverse cardiac events (MACE) during follow-up as the gold standard. RESULTS: The study included 159 patients (median age at functional testing 48 years, median age at HL diagnosis 27 years, 62.9% female). Abnormal functional testing had the highest specificity (94.2% (95% CI 88.4%-97.6%)) and positive likelihood ratio (4.55 (95% CI 1.86-11.13)) while CAC had the highest sensitivity (63.2% (95% CI 46.0%-78.2%)) and lowest negative likelihood ratio (0.52 (95% CI 0.34-0.80)). Specificity for ACC/AHA risk assessment was also high (88.5% (95% CI 81.1%-93.7%)). Over 3.3 years of follow-up, abnormal functional testing (adjusted subdistribution hazard ratio (SHR) 5.10, 95% CI 2.41 - 10.78, p < 0.001) and CAC (adjusted SHR 3.58, 95% CI 1.35 - 9.47, p = 0.010) were both significantly associated with MACE. CONCLUSIONS: In HL survivors treated with RT, both abnormal functional testing and ACC/AHA risk assessment had high specificity for subsequent MACE, but CAC had higher sensitivity. Further research is needed to inform CAD screening and primary prevention strategies in this population.

Role of Exercise Treadmill Testing in the Assessment of Coronary Microvascular Disease.

OBJECTIVES: The authors aimed to study the sensitivity and specificity of exercise treadmill testing (ETT) in the diagnosis of coronary microvascular disease (CMD), as well as the prognostic implications of ETT results in patients with CMD. BACKGROUND: ETT is validated to evaluate for flow-limiting coronary artery disease (CAD), however, little is known about its use for evaluating CMD. METHODS: We retrospectively studied 249 consecutive patients between 2006 and 2016 who underwent ETT and positron emission tomography within 12 months. Patients with obstructive CAD or left ventricular systolic dysfunction were excluded. CMD was defined as a coronary flow reserve <2. Patients were followed for the occurrence of a first major adverse event (composite of death or hospitalization for myocardial infarction or heart failure). RESULTS: The sensitivity and specificity of a positive ETT to detect CMD were 34.7% (95% CI: 25.4%-45.0%) and 64.9% (95% CI: 56.7%-72.5%), respectively. The specificity of a positive ETT to detect CMD increased to 86.8% (95% CI: 80.3%-91.7%) when only classifying studies with ischemic electrocardiogram changes that lasted at least 1 minute into recovery as positive, although at a cost of lower sensitivity (15.3%; 95% CI: 8.8%-24.0%). Over a median follow-up of 6.9 years (IQR: 5.1-8.2 years), 30 (12.1%) patients met the composite endpoint, including 13 (13.3%) with CMD (n = 98). In patients with CMD, ETT result was not associated with the composite endpoint (P = 0.076). CONCLUSIONS: Our data suggest limited sensitivity of ETT to detect CMD. However, a positive ETT with ischemic changes that persist at least 1 minute into recovery in the absence of obstructive CAD should raise suspicion for the presence of CMD given a high specificity. Further study is needed with larger patient sample sizes to assess the association between ETT results and outcomes in patients with CMD.

Urokinase-mediated recruitment of myeloid-derived suppressor cells and their suppressive mechanisms are blocked by MUC1/sec.

The transmembrane isoform of mucin 1 (MUC1/TM) is a well-recognized tumor antigen, contributing to tumorigenesis and immune evasion. Although MUC1/TM has been correlated with malignancy, we have previously reported on antitumor properties and prevention of tumor development by a secreted splice variant of MUC1 (MUC1/sec). Because myeloid-derived suppressor cells (MDSCs) play a critical role in tumor-induced immunosuppression, we investigated their recruitment by tumor cells expressing either MUC1/TM or MUC1/sec. DA-3 tumor cells expressing MUC1/sec recruit dramatically lower levels of MDSCs, relative to MUC1/TM-expressing DA-3 cells. Because MUC1/sec was previously shown to down-regulate tumor expression of urokinase plasminogen activator (uPA), a protease linked to tumor aggressiveness and metastasis, the potential role of uPA in MDSC recruitment was investigated. Tumor-derived uPA is capable of recruiting MDSCs, and correlates with tumor development. In addition to diminishing recruitment of MDSCs, the effect of MUC1/sec on MDSC-suppressive mechanisms was investigated. MUC1/sec, or its unique immunoenhancing peptide, is capable of blocking expression of arginase 1 and production of reactive oxygen species in MDSCs, implicated in the suppression of T cells. These findings demonstrate a new mechanism of MDSC recruitment, and provide evidence that MUC1/sec has antitumor properties affecting MDSCs.

Clinical and Molecular Study of the NOG Gene in Families with Mandibular Micrognathism.

OBJECTIVES: Previous studies showed that noggin gene (NOG) sequence alterations, as well as epigenetic factors, could influence mandibular development. The aim of this study was to analyze clinical characteristics, NOG gene sequences, and promoter methylation sites in patients with mandibular micrognathism. MATERIALS AND METHODS: A total of 35 individuals of five Colombian families were subject to clinical and cephalometric analysis for mandibular micrognathism. One nonaffected individual of each family was included as a control. DNA was isolated from whole blood sample from all individuals by salting out method. Nine NOG gene fragments were amplified by polymerase chain reaction (PCR) and sequenced. Identification of CpG islands for methylation analysis at the NOG gene promoter was performed by MSP-PCR kit (Qiagen R). STATISTICAL ANALYSIS: A descriptive statistical analysis was carried out evaluating the presence or absence of genetics variants and the methylation sites in the NOG gene. RESULTS: NOG sequence results of affected individuals with mandibular micrognathism for one of the families studied demonstrated that they were heterozygous for 672 C/A (new mutation). For a second family, individuals were heterozygous for 567 G/C (single nucleotide polymorphism [SNP] RS116716909). For DNA analyzed from all patients studied, no methylations were observed at the NOG gene promoter region. CONCLUSION: Our results suggested that 672 C/A and 567 G/C variants could be involved in the presence of mandibular micrognathism. Moreover, lack of methylation sites at the NOG gene promoter region of all individuals studied suggests possibly other epigenetic factors could modulate mandibular growth. The search of genetic variants related with mandibular micrognathism will allow to predict in an integral way the development patterns of the patients and therefore establish a better clinical treatment.

Downregulation of interleukin-7 and hepatocyte growth factor in the thymic microenvironment is associated with thymus involution in tumor-bearing mice.

During mammary tumorigenesis, there is a profound thymic involution associated with severe depletion of the most abundant subset of thymocytes, CD4(+)CD8(+) immature cells, and an early arrest in at least two steps of T cell differentiation. Thymic atrophy that is normally related with aging has been observed in other model systems, including graft-vs-host disease (GVHD) and tumor development. However, the mechanisms involved in this phenomenon remain to be elucidated. Vascular endothelial growth factor (VEGF) has been associated with thymic involution, when expressed at high levels systemically. In thymuses of D1-DMBA-3 tumor-bearing mice, this growth factor is diminished relative to the level of normal thymuses. Interestingly, the expression of hepatocyte growth factor (HGF), which has been associated with proliferation, cell survival, angiogenesis and B-cell differentiation, is profoundly down-regulated in thymuses of tumor bearers. In parallel, IL-7 and IL-15 mRNA, crucial cytokines involved in thymocytes development and cellular homeostasis, respectively, are also down-regulated in the thymuses of tumor hosts as compared to those of normal mice. Injection of HGF into mice implanted with mammary tumors resulted in normalization of thymic volume and levels of VEGF, IL-7 and IL-15. While, injections of IL-7 partially restored the thymic involution observed in the thymuses of tumor-bearing mice, injection of IL-15 did not have any significant effects. Our data suggest that the downregulation of HGF and IL-7 may play an important role in the thymic involution observed in tumor-bearing hosts.

Impaired thymopoiesis occurring during the thymic involution of tumor-bearing mice is associated with a down-regulation of the antiapoptotic proteins Bcl-XL and A1.

The thymus is a central lymphoid organ in which T lymphocytes undergo differentiation and maturation without the need for antigenic stimulation. Apoptosis (programmed cell death), plays a critical role in shaping the T cell repertoire, deleting unproductive as well as potentially autoreactive T cells. Thymic atrophy has been observed in several model systems, including aging, graft-vs-host-disease and tumor development. However, the mechanisms involved in this phenomenon remain to be completely elucidated. We have previously shown that the progressive growth of D1-DMBA-3 mammary tumor leads to extreme thymic atrophy in the host. This thymic involution is associated with an early block in T cell maturation at the triple negative stage of differentiation. In the present study we have used our murine mammary tumor model to further analyze the specific T cell subpopulations present in the thymus of tumor-bearing animals as well as to characterize the alterations of the apoptotic process present during the impaired thymopoiesis associated to this thymic involution. Flow cytometric analysis revealed a moderate increase in the percentages of single positive CD4+ and CD8+ cells within the CD3 negative population in the thymuses of tumor-bearing mice. Moreover, we observed a prolonged increase in apoptosis among thymocytes from tumor-bearing mice compared with thymocytes from normal mice during tumor development. Lastly, we found a major decrease of Bcl-XL and A1, two crucial anti-apoptotic Bcl-2 family members that are developmentally regulated in T cells. Together, our data suggest that the severe thymic involution seen in mammary tumor bearers is due to an arrest in at least two steps of T cell differentiation and a down-regulation of important molecules that control programmed cell death.

Decreased levels of both Stat1 and Stat3 in T lymphocytes from mice bearing mammary tumors.

BACKGROUND: Lymphocytes from tumor-bearing animals have been shown to lack antitumor function. The objective of this study was to investigate the status of the signal transducers, Stat1 and Stat3, in T lymphocytes of animals bearing D1-DMBA-3 mammary tumors and to elucidate if any alterations in these signal transducers can be explained by the presence of tumor-derived factors and correlated with the lack of antitumor function in these cells. MATERIALS AND METHODS: T Lymphocytes from spleens of normal and tumor-bearing mice were purified and assayed for the presence of Stat1 and Stat3 by Western blot analysis. RESULTS: It was found that levels of both Stat1 and Stat3 were reduced in T lymphocytes of tumor-bearers not only in their active, phosphorylated form but in total protein levels. CONCLUSION: These findings indicate that during mammary tumor progression, alteration of various transcription factors may contribute to the down-regulation of immune function.

Involvement of protein kinase C and not of NF kappa B in the modulation of macrophage nitric oxide synthase by tumor-derived phosphatidyl serine.

Nitric oxide (NO) is one of the main cytotoxic effector molecules involved in the killing of tumor cells by macrophages. In macrophages, lipopolysaccharide (LPS) alone or in combination with IFN-gamma causes the generation of NO by an inducible form of NO synthase (iNOS). We have previously reported that macrophages from mammary tumor bearers have a downregulation of their NO production leading to a diminished cytotoxic activity. Further studies lead to the isolation and characterization of phosphatidyl serine (PS) as a NO inhibitory factor produced by mammary tumor cells. Pretreatment of macrophages with PS was shown to downregulate their cytotoxic potential and NO production upon stimulation with LPS. Activation of PS-pretreated macrophages with LPS and IFN-gamma resulted in higher levels of NO than those observed with LPS alone, but lower than those of untreated macrophages activated with LPS and IFN-gamma. These results correlated with the levels of iNOS RNA as detected by Northern blot analyses. A study of the expression and binding activity of the transcription factor NF kappa B in macrophages pretreated with PS revealed no differences with untreated macrophages. Investigation of the possible signaling pathways leading to the induction of iNOS revealed that in LPS-stimulated macrophages, increases in internal calcium concentration [Ca2+]i were not observed, while NO was normally produced even under calcium-deprived conditions. In contrast, an effective synergism of IFN-gamma with LPS in the production of NO by macrophages required an optimal increase in [Ca2+]i stimulated by IFN-gamma. This increment in [Ca2+]i was significantly reduced in PS-pretreated macrophages. Further experiments demonstrated that pretreatment of macrophages with PS did not change the normal pattern of tyrosine phosphorylation stimulated by LPS but strikingly inhibited PKC activity. Combinations of LPS and IFN-gamma did not alter the latter result, suggesting that IFN-gamma enhances LPS-induction of iNOS through a pathway other than activation of PKC. Importantly, expression of PKC isozymes in both untreated and PS-pretreated macrophages stimulated with LPS remained constant. Out data suggest that, in tumor bearers, PKC and not NF kappa B is the main target for PS to exert its NO inhibitory action on LPS-activated macrophages. An excess of PS in PS-PKC interaction may be responsible, at least in part, for this type of PKC inhibition. Furthermore, PS also appears to downregulate the rise in [Ca2+]i promoted by IFN-gamma in macrophages, reducing the synergism of this cytokine with LPS and leading to a less effective production of NO.

Immune modulation by melanoma-derived factors.

Melanomas, while the less common of skin cancers, are highly aggressive and once they metastasize usually indicate a poor prognosis. Melanomas are in many cases immunogenic and thus have been a prime target for immunotherapy, which has resulted in objective responses in some patients. To understand why antitumor immunity fails, and for the purpose of discovering new targets to improve therapy, there has been great interest to analyse the antitumor immune responses which exist in these patients, and uncover mechanisms which block tumor-specific immune responses. It is now evident that immunosuppressive cell networks and factors play a major role in the failure of the antitumor immune responses and therapies to eradicate the tumor. In this review, the factors produced by melanomas which can modulate and enhance these suppressive mechanisms are discussed. The roles of immature dendritic cells, neutrophils, T-regulatory cells, myeloid-derived suppressor cells and M2 macrophages or tumor-associated macrophages are described. Furthermore, taking into consideration of the cross-talk which exists among these different cell types and the cycle of immunosuppression which is evident in melanoma cancer patients and animal models, will be important for future therapeutic approaches.

Using Intervention Mapping to Develop and Adapt Two Educational Interventions for Parents to Increase HPV Vaccination Among Hispanic Adolescents.

Introduction: Effective interventions to increase HPV vaccination are needed to reach national vaccination goals and to reduce later HPV-related cancer disparities. We used Intervention Mapping (IM) to develop and adapt a theory- and evidence-based educational intervention targeting parents of Hispanic adolescents to increase HPV vaccination. Methods: We followed IM steps 1-6 to: (1) develop a logic model and identify modifiable factors associated with vaccination among Hispanic adolescents by conducting literature reviews, focus groups, and in-depth interviews with Hispanic parents; (2) develop outcomes, write performance objectives, and develop a matrix of change objectives; (3) develop and identify a program theme, program components, theoretical methods, and practical applications; (4) develop an intervention design plan; (5) develop implementation strategies; and (6) develop an evaluation plan. We completed Steps 1-6 for to develop an intervention targeting parents of females, and we followed the steps again to adapt the program once HPV vaccine recommendations included males. Results: The program Por Nuestras Hijas (For Our Daughters) included two components: a print fotonovela and a tailored interactive multimedia intervention (TIMI). The program utilized the methods tailoring, targeting, framing, anticipated regret, modeling, skill building, and education and counseling to target the following determinants: parental knowledge, attitudes, self-efficacy, skills, perceived benefits/barriers, perceived susceptibility, perceived norms, and outcome expectations as modifiable factors influencing HPV vaccination. Lay health workers implemented the program in community clinics. A logic model of change guided evaluation planning. We later adapted the outcome and intervention content for parents of Hispanic adolescent males and changed the theme to Por Nuestros Hijos (For Our Children). Throughout the development and adaptation processes, we relied on theory, empirical evidence, and new data to make decisions. Discussion: IM provided a systematic methodology for program development and adaptation. Tasks in each step built upon one another integrating findings from the literature, previous research, qualitative findings, and theory to develop two educational programs for parents to increase HPV vaccination. The systematic process allowed us to develop messages and materials targeting factors beyond HPV knowledge or awareness to create behavior change.

Initiation of inflammatory tumorigenesis by CTLA4 insufficiency due to type 2 cytokines.

Genetically predisposed CTLA4 insufficiency in humans is associated with gastric cancer development, which is paradoxical to the prototypical role of CTLA4 in suppressing antitumor immunity. CTLA4 is a critical immune checkpoint against autoimmune disorders. Autoimmunity has been implicated in protumor or antitumor activities. Here, we show that CTLA4 insufficiency initiates de novo tumorigenesis in the mouse stomach through inflammation triggered by host-intrinsic immune dysregulation rather than microbiota, with age-associated progression to malignancy accompanied by epigenetic dysregulation. The inflammatory tumorigenesis required CD4 T cells, but not the TH1 or TH17 subsets. Deficiencies in IL-4 and IL-13 or IL-4 receptor α broke the link between inflammation and initiation of tumorigenesis. This study establishes the causality of CTLA4 insufficiency in gastric cancer and uncovers a role of type 2 inflammation in initiating gastric epithelial transformation. These findings suggest possible improvement of immune therapies by blocking tumorigenic type 2 inflammation while preserving antitumor type 1 immunity.

Diminished expression of transcription factors nuclear factor kappaB and CCAAT/enhancer binding protein underlies a novel tumor evasion mechanism affecting macrophages of mammary tumor-bearing mice.

Interactions between malignant tumors and the host immune system shape the course of cancer progression. The molecular basis of such interactions is the subject of immense interest. Proinflammatory cytokines produced by macrophages are critical mediators of immune responses that contribute to the control of the advancement of neoplasia. We have shown that the expressions of interleukin 12 (IL-12) and inducible nitric oxide synthase (iNOS) are decreased in macrophages from mammary tumor-bearing mice. In this study, we investigated the causes of IL-12 dysregulation and found deficient nuclear factor kappaB (NFkappaB) and CCAAT/enhancer binding protein (C/EBP) expression and function in tumor bearers' peritoneal macrophages. The constitutive expressions of NFkappaB p50, c-rel, p65, and C/EBPalpha and beta, as well as the lipopolysaccharide-induced nuclear translocation and DNA binding of NFkappaB components and C/EBPalpha and beta, are profoundly impaired in macrophages from mice bearing D1-DMBA-3 tumors. Because similar findings occur with the iNOS gene, it seems that it represents a novel mechanism by which tumor-derived factors interfere with the host immune defenses.

A unique mucin immunoenhancing peptide with antitumor properties.

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.

Adipocyte and leptin accumulation in tumor-induced thymic involution.

Cell-mediated immunity is an important defense mechanism against pathogens and developing tumor cells. The thymus is the main lymphoid organ involved in the formation of the cell-mediated immune response by the maturation and differentiation of lymphocytes that travel from the bone marrow, through the lymphatic ducts, to become T lymphocytes. Thymic involution has been associated with aging; however, other factors such as obesity, viral infection and tumor development have been shown to increase the rate of shrinkage of this organ. The heavy infiltration of adipocyte fat cells has been reported in the involuted thymuses of aged mice. In the present study, the possible accumulation of such cells in the thymus during tumorigenesis was examined by immunohistochemistry. A significant number of adipocytes around and infiltrating the thymuses of tumor-bearing mice was observed. Leptin is a pro-inflammatory adipocytokine that enhances thymopoiesis and modulates T cell immune responses. The levels of leptin and adiponectin, another adipocytokine that has anti-inflammatory properties, were examined by western blot analysis. While no changes were observed in the amounts of adiponectin present in the thymuses of the normal and tumor-bearing mice, significantly higher levels of leptin were detected in the thymocytes of the tumor-bearing mice. This correlated with an increase in the expression of certain cytokines, such as interleukin (IL)-2, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF). The co-culture of thymocytes isolated from normal mice with ex vivo isolated adipocytes from tumor-bearing mice yielded similar results. Our findings suggest that the infiltration and accumulation of adipocytes in the thymuses of tumor-bearing mice play an important role in their altered morphology and functions.

Diminished PKC activity and decreased binding of transcription factors are involved in the impaired production of nitric oxide by macrophages from tumor-bearing mice.

In previous studies we have shown that peritoneal macrophages (PEM) from mammary tumor-bearing BALB/c mice (T-PEM) display a diminished ability to lyse tumor cells upon stimulation with LPS, a phenomenon that is associated to a lower production of nitric oxide, and that is reverted upon costimulation with IFN-gamma. The reduced lytic activity and NO production displayed by T-PEM upon LPS activation were earlier shown by us to be due to a diminished transcription of the inducible nitric oxide synthase (iNOS) gene. In the present study, we have investigated the participation of possible signaling molecules and transcription factors - PKC, NF-kappaB, C/EBP and IRF-1 - in the downregulation of NO production in LPS-activated T-PEM. It was found that PKC activity was greatly reduced in T-PEM as compared to normal macrophages, and did not respond to activation. Interestingly, the different PKC isozyme levels were not significantly altered in T-PEM, with the exception of PKC delta. Alterations in the binding activity of the transcription factors NF-kappaB and C/EBP appeared to be involved in the reduced transcription of iNOS previously observed in T-PEM after LPS activation. These results provide evidence that reductions in iNOS transcription secondary to alterations in cell signaling may be responsible for the diminished capacity of macrophages of LPS-activated tumor-bearers to produce NO and lyse tumor targets.