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T cell trafficking at vascular sites has emerged as a key step in antitumor immunity. Chemokines are credited with guiding the multistep recruitment of CD8+ T cells across tumor vessels. However, the multiplicity of chemokines within tumors has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signaling at effector sites. Here, we investigate the hierarchy of chemokine receptor requirements during T cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for GαI-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8+ effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8+ effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumor vascular interface as a critical checkpoint to effective T cell-based cancer immunotherapy.

Background: Surgery, chemotherapy, and radiation often have limited utility for advanced metastatic disease in the liver, and despite its promising activity in select cancers, PD-1 blockade therapy similarly has minimal benefit in this setting. Curaxin, CBL0137, is an experimental anti-cancer drug that disrupts the binding of DNA to histones, destabilizes chromatin, and induces Z-DNA formation which may stimulate anti-tumor immune responses., Methods: Murine cell lines of colon (CT26) and breast (4T1) cancer were interrogated for survival and CBL0137-associated DNA changes in vitro. Immunocompetent models of liver metastases followed by CBL0137 hepatic arterial infusion (HAI) were used to examine in vivo tumor cell DNA alterations, treatment responses, and the immune contexture associated with CBL0137, both alone and in combination with anti-PD-1 therapy., Results: CBL0137 induced immediate changes to favor tumor cell death in vitro and in vivo with an efficient tumor uptake via the HAI route. Toxicity to CBL0137 was minimal and anti-tumor treatment effects were more efficient with HAI compared to intravenous delivery. Immune effects were pronounced with CBL0137 HAI with concurrent depletion of a specific population of myeloid-derived suppressor cells and maintenance of effector T cell populations., Conclusions: Combination of CBL0137 HAI with PD-1 blockade improved survival in 4T1 tumors but not in CT26 tumors, and therapeutic efficacy relies on the finding of simultaneous and targeted depletion of myeloid-derived suppressor cells and skewing of T cell populations to produce synergy with PD-1 blockade therapy.

Objectives: Standard chemotherapy agents, including carboplatin, have known immunogenic properties. We sought to determine how carboplatin may influence lymphocyte trafficking to tumor sites., Methods: Murine models of ovarian cancer were utilized to examine lymphocyte trafficking with common clinically used agents including carboplatin, anti-PD-1 antibody, or anti-VEGFR-2 antibody. Adhesion interactions of lymphocytes with tumor vasculature were measured using intravital microscopy, lymphocyte homing with immunohistochemistry, and treatment groups followed for overall survival., Results: Carboplatin chemotherapy profoundly alters the tumor microenvironment to promote lymphocyte adhesive interactions with tumor vasculature and resultant improvement in lymphocyte trafficking. The measured results seen with carboplatin in the tumor microenvironment were superior to anti-PD-1 treatment or anti-VEGFR-2 which may have contributed to increased overall survival in carboplatin treated groups., Conclusions: These novel findings suggest a role for chemotherapeutic agents to broadly influence anti-tumor immune responses beyond the induction of immunogenic tumor cell death., (Copyright © 2022. Published by Elsevier Inc.)

Background: CIMAvax-EGF is an epidermal growth factor (EGF)-depleting immunotherapy which has shown survival benefit as a switch maintenance treatment after platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC). The primary objective of this trial is to establish the safety and recommended phase II dose (RP2D) of CIMAvax-EGF in combination with nivolumab as second-line therapy for NSCLC., Methods: Patients with immune checkpoint inhibitor-naive metastatic NSCLC were enrolled using a "3+3" dose-escalation design. Toxicities were graded according to CTCAE V4.03. Thirteen patients (one unevaluable), the majority with PD-L1 0%, were enrolled into two dose levels of CIMAvax-EGF., Findings: The combination was determined to be safe and tolerable. The recommended phase 2 dose of CIMAvax-EGF was 2.4 mg. Humoral response to CIMAvax-EGF was achieved earlier and in a greater number of patients with the combination compared to historical control. Four out of 12 evaluable patients had an objective response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Evans, Lee, Wallace, Reid, Muhitch, Dozier, Mesa, Luaces, Santos-Morales, Groman, Cedeno, Cinquino, Fisher, Puzanov, Opyrchal, Fountzilas, Dai, Ernstoff, Attwood, Hutson, Johnson, Mazorra, Saavedra, Leon, Lage, Crombet and Dy.)

Background: Vascularized composite tissue allotransplantation (VCA) to replace limbs or faces damaged beyond repair is now possible. The resulting clear benefit to quality of life is a compelling reason to attempt this complex procedure. Unfortunately, the high doses of immunosuppressive drugs required to protect this type of allograft result in significant morbidity and mortality giving rise to ethical concerns about performing this surgery in patients with non-life-threatening conditions. Here we tested whether we could suppress anti-graft immune activity by using a safe β 2 -adrenergic receptor (AR) agonist, terbutaline, to mimic the natural immune suppression generated by nervous system-induced signalling through AR., Methods: A heterotopic hind limb transplantation model was used with C57BL/6 (H-2b) as recipients and BALB/c (H-2d) mice as donors. To test the modulation of the immune response, graft survival was investigated after daily intraperitoneal injection of β 2 -AR agonist with and without tacrolimus. Analyses of immune compositions and quantification of pro-inflammatory cytokines were performed to gauge functional immunomodulation. The contributions to allograft survival of β 2 -AR signalling in donor and recipient tissue were investigated with β 2 -AR -/- strains., Results: Treatment with the β 2 -AR agonist delayed VCA rejection, even with a subtherapeutic dose of tacrolimus. β 2 -AR agonist decreased T-cell infiltration into the transplanted grafts and decreased memory T-cell populations in recipient's circulation. In addition, decreased levels of inflammatory cytokines (IFN-γ, IL-6, TNF-α, CXCL-1/10 and CCL3/4/5/7) were detected following β 2 -AR agonist treatment, and there was a decreased expression of ICAM-1 and vascular cell adhesion molecule-1 in donor stromal cells., Conclusions: β 2 -AR agonist can be used safely to mimic the natural suppression of immune responses, which occurs during adrenergic stress-signalling and thereby can be used in combination regimens to reduce the dose needed of toxic immunosuppressive drugs such as tacrolimus. This strategy can be further evaluated for feasibility in the clinic., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.

Aberrancies in the tumor microvasculature limit the systemic delivery of anticancer agents, which impedes tumor response. Using human intravital microscopy (HIVM), we hypothesized that HIVM would be feasible in patients with peritoneal carcinomatosis (PC). During cytoreductive surgery with hyperthermic intraperitoneal chemotherapy for PC, HIVM was performed in both tumor and non-tumor areas. The primary outcome was HIVM feasibility to measure vessel characteristics. We secondarily evaluated associations between HIVM vessel characteristics and oncologic outcomes (RECIST response to neoadjuvant therapy and disease-specific survival). Thirty patients with PC were enrolled. Nineteen patients (63.3%) received neoadjuvant therapy. HIVM was feasible in all patients. Compared to non-tumor (control) areas, PC areas had a lower density of functional vessels, higher proportion of non-functional vessels, smaller lumenal diameters, and lower blood flow velocity. Qualitative differences in these vessel characteristics were observed among patients who had partial response, stable disease, or progressive disease after receiving neoadjuvant therapy. However, no statistically significant relationships were found between HIVM vessel characteristics and oncologic outcomes. These novel findings comprise the first-in-human, real-time evidence of the microscopic differences between normal and tumor-associated vessels and form the basis for our larger, ongoing clinical trial appropriately powered to determine the clinical utility of HIVM (NCT03823144).

Despite advances in therapy for melanoma, heterogeneous responses with limited durability represent a major gap in treatment outcomes. The purpose of this study was to determine whether alteration in tumor blood flow could augment drug delivery and improve antitumor responses in a regional model of melanoma. This approach to altering tumor blood flow was termed "dynamic control." Dynamic control of tumor vessels in C57BL/6 mice bearing B16 melanoma was performed using volume expansion (saline bolus) followed by phenylephrine. Intravital microscopy (IVM) was used to observe changes directly in real time. Our approach restored blood flow in non-functional tumor vessels. It also resulted in increased chemotherapy (melphalan) activity, as measured by formation of DNA adducts. The combination of dynamic control and melphalan resulted in superior outcomes compared to melphalan alone (median time to event 40.0 vs 25.0 days, respectively, p = 0.041). Moreover, 25% (3/12) of the mice treated with the combination approach showed complete tumor response. Importantly, dynamic control plus melphalan did not result in increased adverse events. In summary, we showed that dynamic control was feasible, directly observable, and augmented antitumor responses in a regional model of melanoma. Early clinical trials to determine the translational feasibility of dynamic control are ongoing.

Background: Woodchucks (Marmota monax) are a well-accepted animal model for the investigation of spontaneous hepatocellular carcinoma (HCC). As HCC tumors obtain nutrient blood supply exclusively from the hepatic artery, hepatic artery infusion (HAI) has been applied to HCC. However, there is a scarcity of experimental animal models to standardize drug regimens and examine novel agents. The purpose of this study was to establish an HAI model in woodchucks., Materials and Methods: HAI ports were placed in the gastroduodenal artery (GDA) of 11 woodchucks. The ports were infused with either a vehicle (dextrose 5% in water) or an experimental drug, CBL0137, once a week for 3 wk. Technical success rates, anatomical variation, morbidity and mortality, and tumor responses between groups were analyzed., Results: The GDA access was feasible and reproducible in all woodchucks (11/11). The average operation time was 95 ± 20 min with no increase in the levels of liver enzymes detected from either infusate. The most common morbidity of CBL0137 therapy was anorexia after surgery. One woodchuck died due to hemorrhage at the gallbladder removal site from hepatic coagulopathy. Significantly higher CBL0137 concentrations were measured in the liver compared with blood after each HAI. Tumor growth was suppressed after multiple CBL0137 HAI treatments which corresponded to greater T cell infiltration and increased tumor cell apoptosis., Conclusions: HAI via GDA was a feasible and reproducible approach with low morbidity and mortality in woodchucks. The described techniques serve as a reliable platform for the identification and characterization of therapeutics for HCC., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.

The multikinase inhibitor sorafenib is the only standard first-line therapy for hepatocellular carcinoma (HCC). Here, we report the dose-dependent effects of sorafenib on the immune response, which is related to nuclear factor of activated T cells 1 (NFAT1) activity. In vitro and in vivo experiments were performed with low and high doses of sorafenib using human T cells and spontaneous developed woodchuck HCC models. In vitro studies demonstrated that following exposure to a high dose of sorafenib the baseline activity of NFAT1 in T cells was significantly increased. In a parallel event, high dose sorafenib resulted in a significant decrease in T cell proliferation and increased the proportion of PD-1 expressing CD8+ T cells with NFAT1 activation. In the in vivo model, smaller tumors were detected in the low-dose sorafenib treated group compared to the placebo and high-dose treated groups. The low-dose sorafenib group showed a significant tumor growth delay with significantly more CD3+ cells in tumor. This study demonstrates that sorafenib has immunomodulatory effects in a dose- and time-dependent manner. Higher dose of sorafenib treatment was associated with immunosuppressive action. This observed effect of sorafenib should be taken into consideration in the selection of optimum starting dose for future trials.

Intravital microscopy (IVM) is a dynamic imaging modality that allows for the real time observation of biologic processes in vivo , including angiogenesis and immune cell interactions. In the setting of preclinical cancer models, IVM has facilitated an understanding of the tumor associated vasculature and the role of effector immune cells in the tumor microenvironment. Novel approaches to apply IVM to human malignancies have thus far focused on cancer diagnosis and tumor vessel characterization, but have the potential to provide advances in the field of personalized medicine by identifying individual patients who may respond to systemically delivered chemotherapeutic drugs or immunotherapeutic agents. In this review, we highlight the role that IVM has had in investigating tumor vasculature and the tumor microenvironment in preclinical studies and discuss its current and future applications to directly observe human tumors., Competing Interests: CONFLICTS OF INTEREST None.

Background: Vascularized composite tissue allotransplantation (VCA) from a cadaveric donor has now become a clinical reality and the treatment modality of choice for patients with devastating injuries, deformities, and complex tissue defects. However, many VCA patients experience severe toxicities due to the strong immunosuppression required secondary to high antigenicity of the grafts. To improve immunosuppressive protocols for VCA, feasible and reliable preclinical models are necessary. The purpose of this study was to introduce new techniques to an established preclinical VCA model to accelerate future investigations., Methods: C57BL/6 (H-2 b ) and BALB/c (H-2 d ) mice were used to perform VCA as recipients and donors, respectively. Surgery time, success rate, associated complications, and mortality were analyzed. Blood flow in grafts was interrogated with laser speckle image (LSI)., Results: A nonsuture cuff technique was used with the abdominal aorta for end-to-end anastomosis. The cuff technique demonstrated efficiency for donor surgery (52 ± 10 minutes for donor vs. 45 ± 8 minutes for recipient surgery). Successful revascularization was achieved in 27 (90%) of 30 transplants. The majority of surgical complications occurred within 48 hours including artery occlusion, venous occlusion, cerebral stroke, and minor bleeding without mortality. LSI was useful in detecting intraoperative vascular complications with display patterns predictive of complication type., Conclusions: The described techniques may facilitate a more efficient heterotopic hind limb transplantation mouse model of VCA., Competing Interests: The authors declare no conflicts of interest.

Background: The liver has unique anatomy in that most blood flow to normal hepatocytes is derived from the portal venous system, whereas liver tumors obtain their nutrient blood supply exclusively from the hepatic artery. The focused arterial delivery of anticancer agents to liver tumors has been performed for decades; however, preclinical models to standardize drug regimens and examine novel agents have been lacking. The purpose of this study was to establish preclinical hepatic artery infusion (HAI) models in a mouse and to evaluate the safety and delivery capability of the models., Material and Methods: C57BL/6 and BALB/c mice were used to develop models of HAI via the hepatic artery (HA), superior pancreaticoduodenal artery (SPDA), or lienogastric artery (LGA). Success rates, distribution of perfusion, and associated morbidity and mortality were analyzed between groups., Results: All three models were feasible and reproducible in mice, and there was no statistical difference on body weight change between models. The HA model had a 13.3% mortality from acute liver failure, and the SPDA model demonstrated duodenal and pancreatic toxicity. SPDA and LGA routes had the highest success rates (96.7% and 91.4%, respectively) with low mortality, better drug delivery, and preserved physiologic liver function compared with the HA model., Conclusions: The optimal route of HAI was mouse breed specific; SPDA access in BALB/c mice, and the LGA access in C57BL/6 mice. The described techniques serve as a reproducible platform for the identification and characterization of therapeutics for diverse metastatic liver tumors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely restricts antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity., Competing Interests: The authors declare that no competing interests exist.

Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment.

Purpose: While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model., Experimental Design: Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma., Results: Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity., Conclusion: Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.

Nod-like receptors (NLRs) are cytoplasmic pattern recognition receptors that are promising targets for the development of anti-inflammatory therapeutics. Drug discovery efforts targeting NLRs have been hampered by their inherent tendency to form aggregates making protein generation and the development of screening assays very challenging. Herein we report the results of an HTS screen of NLR family member NLRP1 (NLR family, pyrin domain-containing 1) which was achieved through the large scale generation of recombinant GST-His-Thrombin-NLRP1 protein. The screen led to the identification of a diverse set of ATP competitive inhibitors with micromolar potencies. Activity of these hits was confirmed in a FP binding assay, and two homology models were employed to predict the possible binding mode of the leading series and facilitate further lead-optimization. These results highlight a promising strategy for the identification of inhibitors of NLR family members which are rapidly emerging as key drivers of inflammation in human disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fever is a cardinal response to infection that has been conserved in warm-blooded and cold-blooded vertebrates for more than 600 million years of evolution. The fever response is executed by integrated physiological and neuronal circuitry and confers a survival benefit during infection. In this Review, we discuss our current understanding of how the inflammatory cues delivered by the thermal element of fever stimulate innate and adaptive immune responses. We further highlight the unexpected multiplicity of roles of the pyrogenic cytokine interleukin-6 (IL-6), both during fever induction and during the mobilization of lymphocytes to the lymphoid organs that are the staging ground for immune defence. We also discuss the emerging evidence suggesting that the adrenergic signalling pathways associated with thermogenesis shape immune cell function.

Purpose: Quinacrine is a relatively non-toxic drug, once given almost exclusively for malaria. However, recent studies show that quinacrine can suppress nuclear factor-κB (NF-κB), and activate p53 signaling. We investigated the anti-cancer effect of quinacrine, using a novel mouse model of isolated limb perfusion (ILP) for extremity melanoma., Method: Female C57BL/6 mice (22-25 g) were injected with B16 melanoma cells (1 × 10(5)) subcutaneously in the distal thigh. After 7 days of tumor establishment, mice were perfused with either PBS, melphalan (90 µg), or quinacrine (3.5 and 4.5 mg) through the superficial femoral artery for 30 min at either 37 or 42 °C in a non-oxygenated circuit. We analyzed morbidity, toxicity, tumor apoptosis, and responses., Results: Melanoma cell death following in vitro exposure to quinacrine was dose and time dependent. A significant decrease in mean tumor volume was observed after perfusion with low-dose and high-dose quinacrine (both P = 0.002) at 37 °C as well as after perfusion with low-dose quinacrine (P = 0.0008) at 42 °C., Conclusion: Quinacrine has demonstrable efficacy against melanoma cells in vitro and in a clinically relevant model of ILP. Further studies to evaluate the optimal conditions for quinacrine usage are warranted.

Background: Approximately 32% of US children are overweight or obese. Restaurant and fast food meals contribute 18% of daily calories for children and adolescents aged 2 to 18 years. Changing children's menus may improve their diets. This case study describes Best Food for Families, Infants, and Toddlers (Best Food FITS), a community-based intervention designed to address childhood obesity. The objective of this study was to improve San Marcos children's access to healthy diets through partnerships with local restaurants, removing sugar-sweetened beverages, decreasing the number of energy-dense entrées, and increasing fruit and vegetable offerings on restaurant menus., Community Context: San Marcos, Texas, the fastest growing US city, has more restaurants and fewer grocery stores than other Texas cities. San Marcos's population is diverse; 37.8% of residents and 70.3% of children are Hispanic. Overweight and obesity rates among school children exceed 50%; 40.3% of children live below the poverty level., Methods: This project received funding from the Texas Department of State Health Services Nutrition, Physical Activity, and Obesity Prevention Program to develop Best Food FITS. The case study consisted of developing a brand, engaging community stakeholders, reviewing existing children's menus in local restaurants, administering owner-manager surveys, collaborating with restaurants to improve menus, and assessing the process and outcomes of the intervention., Outcome: Best Food FITS regularly participated in citywide health events and funded the construction of a teaching kitchen in a new community building where regular nutrition classes are held. Sixteen independent restaurants and 1 chain restaurant implemented new menus., Interpretation: Improving menus in restaurants can be a simple step toward changing children's food habits. The approach taken in this case study can be adapted to other communities. Minimal funding would be needed to facilitate development of promotional items to support brand recognition.

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

Within the tumor microenvironment, IL-6 signaling is generally considered a malevolent player, assuming a dark visage that promotes tumor progression. Chronic IL-6 signaling is linked to tumorigenesis in numerous mouse models as well as in human disease. IL-6 acts intrinsically on tumor cells through numerous downstream mediators to support cancer cell proliferation, survival, and metastatic dissemination. Moreover, IL-6 can act extrinsically on other cells within the complex tumor microenvironment to sustain a pro-tumor milieu by supporting angiogenesis and tumor evasion of immune surveillance. A lesser known role for IL-6 signaling has recently emerged in which it plays a beneficial role, presenting a fairer face that opposes tumor growth by mobilizing anti-tumor T cell immune responses to attain tumor control. Accumulating evidence establishes IL-6 as a key player in the activation, proliferation and survival of lymphocytes during active immune responses. IL-6 signaling can also resculpt the T cell immune response, shifting it from a suppressive to a responsive state that can effectively act against tumors. Finally, IL-6 plays an indispensable role in boosting T cell trafficking to lymph nodes and to tumor sites, where they have the opportunity to become activated and execute their cytotoxic effector functions, respectively. Here, we discuss the dual faces of IL-6 signaling in the tumor microenvironment; the dark face that drives malignancy, and the fairer aspect that promotes anti-tumor adaptive immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cancer immunotherapy aims to generate long-lived, tumour-specific adaptive immunity to limit dysregulated tumour progression and metastasis. Tumour vasculature has emerged as a critical checkpoint controlling the efficacy of immunotherapy since it is the main access point for cytotoxic T cells to reach tumour cell targets. Therapeutic success has been particularly challenging to achieve because of the local, cytokine-rich inflammatory milieu that drives a pro-tumourigenic programme supporting the growth and survival of malignant cells. Here, we focus on recent evidence that systemic thermal therapy can switch the activities of the inflammatory cytokine, interleukin-6 (IL-6), to a predominantly anti-tumourigenic function that promotes anti-tumour immunity by mobilising T cell trafficking in the recalcitrant tumour microenvironment.

Immune cells are key regulators of neoplastic progression, which is often mediated through their release of cytokines. Inflammatory cytokines such as IL-6 exert tumor-promoting activities by driving growth and survival of neoplastic cells. However, whether these cytokines also have a role in recruiting mediators of adaptive anticancer immunity has not been investigated. Here, we report that homeostatic trafficking of tumor-reactive CD8+ T cells across microvascular checkpoints is limited in tumors despite the presence of inflammatory cytokines. Intravital imaging in tumor-bearing mice revealed that systemic thermal therapy (core temperature elevated to 39.5°C ± 0.5°C for 6 hours) activated an IL-6 trans-signaling program in the tumor blood vessels that modified the vasculature such that it could support enhanced trafficking of CD8+ effector/memory T cells (Tems) into tumors. A concomitant decrease in tumor infiltration by Tregs during systemic thermal therapy resulted in substantial enhancement of Tem/Treg ratios. Mechanistically, IL-6 produced by nonhematopoietic stromal cells acted cooperatively with soluble IL-6 receptor-α and thermally induced gp130 to promote E/P-selectin- and ICAM-1-dependent extravasation of cytotoxic T cells in tumors. Parallel increases in vascular adhesion were induced by IL-6/soluble IL-6 receptor-α fusion protein in mouse tumors and patient tumor explants. Finally, a causal link was established between IL-6-dependent licensing of tumor vessels for Tem trafficking and apoptosis of tumor targets. These findings suggest that the unique IL-6-rich tumor microenvironment can be exploited to create a therapeutic window to boost T cell-mediated antitumor immunity and immunotherapy.

An effectively orchestrated immune response to infection and disease depends on efficient trafficking of lymphocytes across vascular beds at distinct tissue sites. Local inflammation and systemic fever increase immune surveillance to immune-relevant sites throughout the body. During the initiation phase of inflammation, this tightly regulated process improves leukocyte trafficking to the secondary lymphoid organs where they undergo activation and expansion in response to cognate antigen. In the resolution phase following the clearance of the invading pathogen, lymphocyte entry is rapidly returned to baseline conditions. Specialized blood vessels termed high endothelial venules (HEVs) have emerged as critical 'hotspots' controlling the rate of lymphocyte entry into lymphoid organs during both phases of inflammation. In this review, we will examine the remarkably tight regulation of lymphocyte trafficking across HEVs conferred by inflammatory cues associated with the thermal element of fever. These studies have revealed a novel role for interleukin-6 (IL-6) trans-signaling in eliciting systemic effects on lymphocyte trafficking patterns to fine-tune immune surveillance.

Objective: Fever is associated with improved survival, although its beneficial mechanisms are poorly understood. Previous studies indicate that the thermal element of fever augments lymphocyte migration across high endothelial venules (HEVs) of lymphoid organs by increasing the intravascular display of a gatekeeper trafficking molecule, intercellular adhesion molecule-1 (ICAM-1). Here, we evaluated the spatio-temporal relationship between the thermal induction of intravascular ICAM-1 and lymphocyte trafficking., Methods: Intravascular ICAM-1 density was quantified by immunofluorescence staining in mice exposed to fever-range whole-body hyperthermia (39.5+/-0.5 degrees C). ICAM-1-dependent lymphocyte trafficking was measured in short-term homing assays., Results: A linear relationship was observed between the duration of heat treatment and intravascular ICAM-1 density in HEVs with maximal responses requiring sustained (i.e., five hours) thermal stress. Circulating lymphocytes were found to sense incremental changes in ICAM-1 on HEVs, such that trafficking is proportional to the intravascular density of ICAM-1. We further identified a hydroxamate-sensitive shedding mechanism that restores ICAM-1 expression to homeostatic levels following the cessation of thermal stress., Conclusions: The time-dependent response to thermal stress indicates that ICAM-1 density governs the efficiency of lymphocyte interactions with HEVs in vivo. These studies highlight the dynamic role of the microcirculation in promoting immune surveillance during febrile inflammatory responses.

Immune protection from microbial invaders or malignant progression is dependent on the ability of lymphocytes to efficiently traffic across morphologically and biochemically distinct vascular sites throughout the body. Lymphocyte trafficking to target tissues is orchestrated by adhesion molecules and chemokines that stabilize dynamic interactions between circulating lymphocytes and endothelial cells lining blood vessels. While the molecular mechanisms that regulate the efficient migration of lymphocytes across specialized high endothelial venules (HEVs) in secondary lymphoid organs have been extensively characterized, there is a paucity of information available regarding the mechanisms that dictate the rate of lymphocyte entry into tumor tissues. This article summarizes recent evidence that inflammatory cues associated with fever-range thermal stress promote lymphocyte extravasation across HEVs of lymphoid organs through a highly regulated lymphocyte-endothelial-interleukin-6 (IL-6) biological axis. The potential for using thermally-based strategies to improve lymphocyte delivery to the tumor microenvironment during T cell-based immunotherapy will also be discussed.

Study Objective: We sought to assess accuracy of visual diagnosis of laparoscopically excised visceral and peritoneal abnormalities suggestive of endometriosis by comparison with final histologic diagnosis., Design: Prospective study of 2005 tissue specimens from 512 patients undergoing laparoscopy for evaluation of pelvic pain was conducted (Canadian Task Force classification II-2)., Setting: A private practice referral center for treatment of endometriosis., Patients: From February 1992 through December 1998, 512 patients underwent laparoscopic excision of endometriosis. In all patients, the primary indication for surgery was pelvic pain with either prior surgical diagnosis or clinical history consistent with endometriosis., Interventions: All areas of viscera and peritoneum either with typical appearance suggestive of endometriosis or atypical appearance were completely excised and examined histologically. At laparoscopy, all excised lesions were documented in a diagram by the primary surgeon according to anatomic site and visual description and were labeled as either suggestive of endometriosis or otherwise atypical in appearance. The hospital pathology department received entire lesions fixed in formalin and evaluated specimens for presence or absence of endometriosis. Pathologists, who were blinded to the surgeon's suspicion of endometriosis, were provided only the anatomic site of excised tissue. By definition, diagnosis of endometriosis was made when histologic evidence existed of both endometrial glands and stroma., Measurements and Main Results: Diagrams detailing appearance, anatomic site, and surgeon's suspicion of endometriosis versus atypical lesions were compared with final histologic diagnosis. The greatest number of patient lesions were excised from cul-de-sac (n = 309). For this site, using visual criteria for diagnosis of endometriosis, positive predictive value was 93.9%, sensitivity was 69.3%, negative predictive value was 41.9%, and specificity was 83.1%. Prevalence was noted to be 79.0% and accuracy was 72.2%. In addition, atypical-appearing tissue not presumed to be endometriosis was confirmed to be endometriosis histologically in 24.3%. In examining tissue specimens from multiple anatomic sites, laparoscopic visual diagnosis of typical endometriosis generally had high positive predictive value. However, both sensitivity and negative predictive value were lower than expected because of atypical lesions subsequently diagnosed as endometriosis., Conclusions: These data suggest that when the surgical objective is complete eradication of endometriosis, the surgeon must be prepared to excise all lesions suggestive of endometriosis and tissue atypical in appearance as in most anatomic sites approximately 25% of atypical specimens proved to be endometriosis.

Objective: This retrospective observational report analyzes the demographics, blood loss, length of surgical duration, number of days in the hospital, and complications for 821 consecutive patients undergoing total laparoscopic hysterectomy over a 11-year period stratified by incidental appendectomy., Methods: A retrospective chart abstraction was performed. ANOVA and chi-square tests were performed with significance preset at P<0.05., Results: Of 821 consecutive patients undergoing total laparoscopic hysterectomy, 257 underwent elective appendectomy with the ultrasonic scalpel, either as part of their staging, treatment for pelvic pain, or prophylaxis against appendicitis. Comparing the 2 groups, no difference existed in mean age of 50+/-10 years or mean BMI of 27.6+/-6.7. Both groups had a similar mean blood loss of 130 mL. Surgery took less time (137 vs 118 minutes, P<0.0012) and the hospital stay was shorter in the appendectomy group (1.5 vs 1.2, P<0.0001) possibly because it was performed incidentally in most cases. No complications were attributable to the appendectomy, and complication types and rates in both groups were similar. Though all appendicies appeared normal, pathology was documented in 9%, including 3 carcinoid tumors., Conclusions: Incidental appendectomy during total laparoscopic hysterectomy is not associated with significant risk and can be routinely offered to patients planning elective gynecologic laparoscopic procedures, as is standard for open procedures.

Study Objective: A key component of hysteroscopic complications, such as fluid overload and severe dilutional hyponatremia, is the failure to anticipate and quickly recognize fluid deficits. The purpose of this study was to measure the volume and mass of irrigation fluid bags to assess the overfill of 3 common types of hysteroscopy irrigation fluids, 0.9% normal saline solution, 3% sorbitol, and 1.5% glycine, to challenge the often-quoted standard of assumption that overfill may be as high as 10% of the bag's volume., Design: Ten cases of irrigation fluid were tested. The volume and weight of drained fluid from 18 bags of 0.9% normal saline solution 2000 mL, 12 bags 3% sorbitol 3000 mL, 8 bags of 1.5% glycine 3000 mL, and 4 bags of 0.9% normal saline solution 5000 mL were measured. Institutional review board exemption was obtained., Measurements and Main Results: Ten cases of irrigation fluid were obtained. The volume and weight of drained fluid from 18 bags of 0.9% normal saline solution 2000 mL, 12 bags of 3% sorbitol 3000 mL, 8 bags of 1.5% glycine 3000 mL, and 4 bags of 0.9% normal saline solution 5000 mL were measured. By volume, varying by the type of fluid tested, the maximum observed overfill was between 3.3% to 5.0%. For confirmation, each bag was also weighed and found to have a maximum overfill between 2.8% to 5.6%, varying with the volume and type of fluid measured. These findings were then compared with the manufacturer-provided overfill range of 1.5% to 6.0%. No underfill was observed., Conclusion: Contrary to assertions over the last 25 years that overfill is 10% or higher as a rule, it appears more reasonable to assume that the degree of overfill is contingent on the type and volume of fluid used and is more likely closer to 2.8% to 5.6%. Therefore an accurate collecting system and weight measurement is more precise.

Fever is an evolutionarily conserved response during acute inflammation, although its physiological benefit is poorly understood. Here we show thermal stress in the range of fever temperatures increased the intravascular display of two 'gatekeeper' homing molecules, intercellular adhesion molecule 1 (ICAM-1) and CCL21 chemokine, exclusively in high endothelial venules (HEVs) that are chief portals for the entry of blood-borne lymphocytes into lymphoid organs. Enhanced endothelial expression of ICAM-1 and CCL21 was linked to increased lymphocyte trafficking across HEVs. A bifurcation in the mechanisms controlling HEV adhesion was demonstrated by evidence that the thermal induction of ICAM-1 but not of CCL21 involved an interleukin 6 trans-signaling pathway. Our findings identify the 'HEV axis' as a thermally sensitive alert system that heightens immune surveillance during inflammation by amplifying lymphocyte trafficking to lymphoid organs.

Objective: We sought to analyze surgical results of women with uterine cancers having TLH+/-staging, stratifying data by body mass index (BMI)., Methods: This is a retrospective analysis of data from 9 years, using Pearson and Spearman correlations, ANOVA and Fisher's Exact Test with significance at P<0.05, stratifying by BMI (kg/m2): underweight (<18.5 kg/m2), ideal (18.5-24.9 kg/m2), overweight (25 to 29.9 kg/m2), obese (30 to 39.9 kg/m2) and morbidly obese (40 kg/m2 or more)., Results: Of 702 patients having TLH over 9 years, 90 patients had uterine pathology. Two (2%) procedures were converted to laparotomy due to unsuspected widespread metastasis and excluded from analysis. BMI ranged from 18 to 60 kg/m2, with 31 patients having ideal, 19 having overweight and 38 having obese BMI. Of these, 19 patients had hyperplasia, while 63 had endometrial carcinoma, 1 had both ovarian and endometrial carcinoma and 5 had sarcoma. Of these 88 patients, 61 had TLH while 27 patients had indicated pelvic and aortic node dissection. The mean age was 60 years, and mean parity was 1.5 for all BMI groups. There were no significant differences in mean duration of surgery (150 min), blood loss (129 cm3) and days in hospital (1.7 days) for all BMI groups. There was no significant difference in uterine weight (140 gm) or number of nodes dissected (21 nodes). Complications occurred in 4 patients (4.5%): 1 diverticulitis, 1 ureteral injury, 1 laparotomy for bleeding and 1 incisional hernia., Conclusions: Total laparoscopic hysterectomy is feasible and safe for women with uterine neoplasia for every BMI category and extends the benefits of minimally invasive hysterectomy to more women, regardless of BMI.

Migration of blood-borne lymphocytes into tissues involves a tightly orchestrated sequence of adhesion events. Adhesion molecules and chemokine receptors on the surface of circulating lymphocytes initiate contact with specialized endothelial cells under hemodynamic shear prior to extravasation across the vascular barrier into tissues. Lymphocyte-endothelial adhesion occurs preferentially in high endothelial venules (HEV) of peripheral lymphoid organs. The continuous recirculation of naïve and central memory lymphocytes across lymph node and Peyer's patch HEV underlies immune surveillance and immune homeostasis. Lymphocyte-endothelial interactions are markedly enhanced in HEV-like vessels of extralymphoid organs during physiological responses associated with acute and chronic inflammation. Similar adhesive mechanisms must be invoked for efficient trafficking of immune effector cells to tumor sites in order for the immune system to have an impact on tumor progression. Here we discuss recent evidence for the role of fever-range thermal stress in promoting lymphocyte-endothelial adhesion and trafficking across HEV in peripheral lymphoid organs. Findings are also presented that support the hypothesis that lymphocyte-endothelial interactions are limited within tumor microenvironments. Further understanding of the molecular mechanisms that dynamically promote lymphocyte trafficking in HEV may provide the basis for novel approaches to improve recruitment of immune effector cells to tumor sites.

An important consideration in the development of T cell-based cancer immunotherapy is that effector T cells must efficiently traffic to the tumor microenvironment in order to control malignant progression. T cell trafficking to target tissues is orchestrated by dynamic interactions between circulating lymphocytes and endothelial cells lining blood vessels. It is informative, in this regard, to compare and contrast the molecular mechanisms governing lymphocyte extravasation at distinct vascular sites: (1) high endothelial venules (HEV) of secondary lymphoid organs, which are portals for efficient trafficking of naive and central memory T lymphocytes; (2) non-activated endothelium of normal tissues that mediate relatively low basal levels of trafficking but are rapidly transformed into HEV-like vessels in response to local inflammatory stimuli; and (3) vessels within the intratumoral region and the surrounding peritumoral areas. These vessels can be distinguished by differential expression of hallmark trafficking molecules that function as molecular beacons directing lymphocyte migration across vascular barriers. This article reviews evidence that recruitment of effector T cells to the intratumoral microenvironment is impeded by sub-threshold expression of trafficking molecules on tumor microvessels. Emerging data support the thesis that when considered from the perspective of extravasation, vessels embedded within the intratumoral microenvironment of established tumors do not exhibit stereotypical characteristics of a chronic inflammatory state. A major challenge will be to develop therapeutic approaches to improve trafficking of effector T lymphocytes to tumor sites without skewing the balance in favor of a chronic inflammatory milieu that facilitates tumor maintenance and progression.

Objective: to describe stressors experienced by women living in an economically disenfranchised urban community and test the relationships between those stressors and women's self-reported health status., Methods: We used a stress process framework to examine the implications of economic divestment and race-based residential segregation on the lives and health of women raising children in Detroit. We conducted qualitative analysis of in-depth interviews with 48 community residents and surveyed 679 women raising children in this community. Regression models controlling for age, education, and income examined the relationships of each of these stressors to symptoms of depression and general health status., Results: Stressors described by women in the in-depth interviews included financial, work, family, safety, police and other municipal services, and disrespect or unfair treatment. Financial, police, and safety stress and unfair treatment were significantly associated with symptoms of depression; financial and family stress were significantly associated with self-reported general health status., Conclusion: Our results support the hypothesis that life stressors associated with economic divestment contribute to the disproportionate burden of disease experienced by African-American women residing in urban communities. Efforts to address racial and socioeconomic disparities in women's health should include policies that support economic development and municipal infrastructure as fundamental to the maintenance of health.

Purpose: Few studies have examined the impact of long-term treatments or exposures on the development of cataract in maturity-onset animal models. We studied the effect of treatment with D-pantethine and exposure to ultraviolet-B (UVB) radiation on the development of lenticular opacity in the Emory mouse., Methods: A total of 164 Emory mice were randomized by litter at weaning to exposure to UVB light at 12 mJ/cm(2) for 6 hr/day (UV) or usual room light (A), and within litter, were further randomized to bi-weekly intra-peritoneal injections of 0.8 g/kg pantethine (T) or no treatment (C). Retro illumination lens photos were taken at 2, 4, 6, 8, and 10 months after weaning, and graded in masked fashion. The animals were sacrificed at 10 months and the lenses analyzed for total pantethine and total cysteamine., Results: Lens pantethine and cysteamine levels were significantly (P < 0.001) higher for the T as compared to C litters. Mean cataract grade increased monotonically over time for all four groups. Unadjusted mean grade for the AT group at 8 (1.32) and 10 (1.86) months appeared lower than for the other groups (AC: 2.17, 2.39; UVC: 1.77, 2.40; UVT: 1.88, 2.37). However, the mean grade for the pantethine-treated litters did not differ significantly from the untreated litters except at 2 months (when untreated litters had significantly lower grades), when adjusting for UV treatment, gender and litter effect. No significant difference in cataract score existed between UV-exposed and ambient litters. Mortality was higher among pantethine-treated (hazard ratio = 1.8, p = 0.05) and UV-exposed animals (hazard ratio = 1.8, p = 0. 03) than among the untreated and unexposed litters., Conclusion: Significantly increased lens levels of pantethine are achieved with long-term intra-peritoneal dosing. The impact of pantethine on the progression of lenticular opacity in the Emory mouse is less than has been reported in other models. This level of chronic UVB exposure appeared to have no effect on the development of cataract in this model.