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Stress correlates with reduced tumor size and burden, VEGF and CCL2 levels, and infiltrating macrophages compared to unstressed mice.

Psychological stressors have been implicated in the progression of various tumor types. We investigated a role for stress in tumor immune cell chemotaxis in the B16F10 mouse model of malignant melanoma. We exposed female mice to 6-hour periods of restraint stress (RST) for 7 days, then implanted B16F10 malignant melanoma tumor cells and continued the RST paradigm for 14 additional days. We determined serum corticosterone and liver catecholamine concentrations in these mice. To evaluate the tumor microenvironment, we performed IHC and examined cytokine expression profiles using ELISA-based analysis of tumor homogenates. We found that tumors in mice subjected to RST grew significantly slower, had reduced tumor C-C motif ligand 2 (CCL2), and contained fewer F4/80-positive macrophages than tumors from unstressed mice. We observed a concomitant increase in norepinephrine among the RST mice. An in vitro assay confirmed that norepinephrine downregulates CCL2 production in both mouse and human macrophages, and that pretreatment with the pan-β-adrenergic receptor inhibitor nadolol rescues this activity. Furthermore, RST had no effect on tumor growth in transgenic CCL2-deficient mice. This study suggests that stress reduces malignant melanoma by reducing recruitment of tumor-promoting macrophages by CCL2.

Neither norepinephrine nor recombinant CCL2 have proliferative effects on B16F10 cells.

Restraint stress increases tumor CD3+/CD4+ T cell infiltration

Neither norepinephrine nor recombinant CCL2 have toxic effects on B16F10 cells.

Stress confers survival in a murine model of melanoma.

Supplementary Table for qPCR sequences

We report an isotopologue of the trityl radical OX071, labeled with 13C at the central carbon highly sensitive to molecular tumbling.

INTRODUCTION/BACKGROUND: This study assessed the safety and systemic (abscopal) response from the addition of local stereotactic body radiation therapy (SBRT) to checkpoint inhibitor (CPI) immunotherapy in patients with metastatic non-small cell lung cancer (NSCLC). PATIENTS/METHODS: Thirty-five patients with at least two sites of measurable disease on PET/CT received standard-of-care CPI immunotherapy alone (n=19), or in combination with 4 cycles doublet carboplatin/pemetrexed chemotherapy (n=16), and 3–5 fractions SBRT to a single extracranial target lesion between cycles 1–2 of the systemic therapy. Adverse events were assessed using CTCAE version 5.0. Best systemic objective response rate (ORR) was assessed using iRECIST criteria, excluding any irradiated lesion(s). Additional SBRT to a different target lesion was offered to patients who continued on immunotherapy with unconfirmed progressive disease or mixed response. RESULTS: Fifteen patients (44%) experienced 22 grade 1–2 toxicities potentially attributable to radiation, most commonly pneumonitis (n=9) and fatigue (n=6), and no grade 3–5 radiation-induced toxicities. Patients undergoing combined CPI-chemotherapy received a lower median biologically effective dose of SBRT than those undergoing CPI monotherapy (43.2 vs. 60Gy), but had a higher rate of radiation-induced toxcity (56% vs. 32%, p

Low-field electron paramagnetic resonance spectroscopy paired with pTAM, a mono-phosphonated triarylmethyl radical, is an unmatched technique for concurrent and non-invasive measurement of oxygen concentration, pH, and inorganic phosphate concentration for in vivo investigations. However, the prior reported synthesis is limited by its low yield and poor scalability, making wide-spread application of pTAM unfeasible. Here, we report a new strategy for the synthesis of pTAM with significantly greater yields demonstrated on a large scale. We also present a standalone application with user-friendly interface for automatic spectrum fitting and extraction of pO2, pH, and [Pi] values. Finally, we confirm that pTAM remains in the extracellular space and has low cytotoxicity appropriate for local injection., We report a new strategy for the synthesis of a mono-phosphonated triarylmethyl radical spin probe and a standalone application with a user-friendly interface for automatic spectrum fitting and extraction of pO2, pH, and [Pi] values.

Psychological stressors have been implicated in the progression of various tumor types. We investigated a role for stress in tumor immune cell chemotaxis in the B16F10 mouse model of malignant melanoma. We exposed female mice to 6-hour periods of restraint stress (RST) for 7 days, then implanted B16F10 malignant melanoma tumor cells and continued the RST paradigm for 14 additional days. We determined serum corticosterone and liver catecholamine concentrations in these mice. To evaluate the tumor microenvironment, we performed IHC and examined cytokine expression profiles using ELISA-based analysis of tumor homogenates. We found that tumors in mice subjected to RST grew significantly slower, had reduced tumor C-C motif ligand 2 (CCL2), and contained fewer F4/80-positive macrophages than tumors from unstressed mice. We observed a concomitant increase in norepinephrine among the RST mice. An in vitro assay confirmed that norepinephrine downregulates CCL2 production in both mouse and human macrophages, and that pretreatment with the pan-β-adrenergic receptor inhibitor nadolol rescues this activity. Furthermore, RST had no effect on tumor growth in transgenic CCL2-deficient mice. This study suggests that stress reduces malignant melanoma by reducing recruitment of tumor-promoting macrophages by CCL2.

This study aimed to determine and compare soft tissue healing outcomes following implant placement in grafted (GG) and non-grafted bone (NGG).Patients receiving single implant in a tooth-bound maxillary non-molar site were recruited. Clinical healing was documented. Volume and content of wound fluid (WF; at 3, 6, and 9 days) were compared with adjacent gingival crevicular fluid (GCF; at baseline, 1, and 4 months). Buccal flap blood perfusion recovery and changes in bone thickness were recorded. Linear mixed model regression analysis and generalized estimating equations with Bonferroni adjustments were conducted for repeated measures.Twenty-five patients (49 ± 4 years; 13 males; nine NGG) completed the study. Soft tissue closure was slower in GG (P 0.01). Differential response in WF/GCF protein concentrations was detected for ACTH (increased in GG only) and insulin, leptin, osteocalcin (decreased in NGG only) at day 6 (P ≤0.04), with no inter-group differences at any time(P 0.05). Blood perfusion rate decreased immediately postoperatively (P 0.01, GG) followed by 3-day hyperemia (P 0.05 both groups). The recovery to baseline values was almost complete for NGG whereas GG stayed ischemic even at 4 months (P = 0.05). Buccal bone thickness changes were significant in GG sites (P ≤ 0.05).History of bone grafting alters the clinical, physiological, and molecular healing response of overlying soft tissues after implant placement surgery.

Clinical parameters available to evaluate early healing phases of bone regeneration procedures are limited. This study explores wound fluid (WF) content for molecular markers to differentiate wound healing responses in the early postoperative period after bone graft placement. Fifteen patients (50 ± 5 years old; 8 men) scheduled to receive tooth extraction and bone graft placement at maxillary nonmolar single-tooth sites were recruited. Primary wound closure was not intended at time of surgery. Gingival crevicular fluid from adjacent teeth or WF from surgical wound edges were collected (30 seconds) at baseline, at 3, 6, and 9 days, and at 1 and 4 months. Multiplex protein assay was used to determine concentration of various wound healing mediators. Immediately after surgery, 87% of surgical sites exhibited open wound. At day 9, mean wound exposure was 4.8 ± 0.4 mm. At 1 month, all wounds were clinically closed. The WF tripled in volume at day 3 and day 6 (P ≤ .05), compared with baseline gingival crevicular fluid, and gradually decreased as wounds closed. The WF concentrations of interleukin (IL)-6, placental growth factor, plasminogen activator inhibitor 1, insulin-like growth factor binding protein 1, and soluble cluster determinant 40 ligand were increased during early healing days, generally with peak concentration at day 6 (P ≤ .004). Conversely, WF concentrations of IL-18 and epidermal growth factor were decreased after surgery, generally not reaching baseline values until wound closure (P ≤ .008). In general, WF cytokine expression kinetics were concordant with wound closure dynamics (P ≤ .04). These results suggest that WF molecular markers such as IL-6, and to a lesser extent placental growth factor and IL-18, might help differentiate wound healing responses after bone regeneration procedures.

Simple Summary The survival rates for patients with pancreatic adenocarcinoma are very low. This dismal prognosis is due in part to late detection and early development of metastases, and successful treatments for pancreatic adenocarcinoma are also lacking. One potential method of treatment is immunotherapy, which has been successfully implemented in several cancers. Despite success in other cancer types, there has been little progress in pancreatic adenocarcinoma. To understand these shortcomings, we explore the roles of macrophages and neutrophils, two prominent immune cell types in the pancreatic tumor environment. In this review, we discuss how macrophages and neutrophils lead to the harsh environment that is unique to pancreatic adenocarcinoma. We further explore how these immune cells can impact standard of care therapies and decrease their effectiveness. Macrophages and neutrophils could ultimately be targeted to improve outcomes for patients with pancreatic adenocarcinoma. Abstract Despite modest improvements in survival in recent years, pancreatic adenocarcinoma remains a deadly disease with a 5-year survival rate of only 9%. These poor outcomes are driven by failure of early detection, treatment resistance, and propensity for early metastatic spread. Uncovering innovative therapeutic modalities to target the resistance mechanisms that make pancreatic cancer largely incurable are urgently needed. In this review, we discuss the immune composition of pancreatic tumors, including the counterintuitive fact that there is a significant inflammatory immune infiltrate in pancreatic cancer yet anti-tumor mechanisms are subverted and immune behaviors are suppressed. Here, we emphasize how immune cell interactions generate tumor progression and treatment resistance. We narrow in on tumor macrophage (TAM) spatial arrangement, polarity/function, recruitment, and origin to introduce a concept where interactions with tumor neutrophils (TAN) perpetuate the microenvironment. The sequelae of macrophage and neutrophil activities contributes to tumor remodeling, fibrosis, hypoxia, and progression. We also discuss immune mechanisms driving resistance to standard of care modalities. Finally, we describe a cadre of treatment targets, including those intended to overcome TAM and TAN recruitment and function, to circumvent barriers presented by immune infiltration in pancreatic adenocarcinoma.

Automation has become an essential component of modern scientific instruments which often capture large amounts of complex dynamic data. Algorithms are developed to read multiple sensors in parallel with data acquisition and to adjust instrumental parameters on the fly. Decisions are made on a time scale unattainable to the human operator. In addition to speed, automation reduces human error, improves the reproducibility of experiments, and improves the reliability of acquired data. An automatic digital control (ADiC) was developed to reliably sustain critical coupling of a resonator over a wide range of time-varying loading conditions. The ADiC uses the computational power of a microcontroller that directly communicates with all system components independent of a personal computer (PC). The PC initiates resonator tuning and coupling by sending a command to MC via serial port. After receiving the command, ADiC establishes critical coupling conditions within approximately 5 ms. A printed circuit board resonator was designed to permit digital control. The performance of the resonator together with the ADiC was evaluated by varying the resonator loading from empty to heavily loaded. For the loading, samples containing aqueous sodium chloride that strongly absorb electromagnetic waves were used. A previously reported rapid scan (RS) electron paramagnetic resonance (EPR) imaging instrument was upgraded by the incorporation of ADiC. RS spectra and an in vivo image of oxygen in a mouse tumor model have been acquired using the upgraded system. ADiC robustly sustained critical coupling of the resonator to the transmission line during these measurements. The design implemented in this study can be used in slow-scan and pulsed EPR with modifications.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a five-year survival rate of only 9%. PDAC is characterized by a dense, fibrotic stroma composed of extracellular matrix (ECM) proteins. This desmoplastic stroma is a hallmark of PDAC, representing a significant physical barrier that is immunosuppressive and obstructs penetration of cytotoxic chemotherapy agents into the tumor microenvironment (TME). Additionally, dense ECM promotes hypoxia, making tumor cells refractive to radiation therapy and alters their metabolism, thereby supporting proliferation and survival. In this review, we outline the significant contribution of fibrosis to the pathogenesis of pancreatic cancer, with a focus on the cross talk between immune cells and pancreatic stellate cells that contribute to ECM deposition. We emphasize the cellular mechanisms by which neutrophils and macrophages, specifically, modulate the ECM in favor of PDAC-progression. Furthermore, we investigate how activated stellate cells and ECM influence immune cells and promote immunosuppression in PDAC. Finally, we summarize therapeutic strategies that target the stroma and hinder immune cell promotion of fibrogenesis, which have unfortunately led to mixed results. An enhanced understanding of the complex interactions between the pancreatic tumor ECM and immune cells may uncover novel treatment strategies that are desperately needed for this devastating disease.

An electron paramagnetic resonance (EPR) imaging system has been custom built for use in pre-clinical and, potentially, clinical studies. Commercial standalone modules have been used in the design that are MATLAB-controlled. The imaging system combines digital and analog technologies. It was designed to achieve maximum flexibility and versatility and to perform standard and novel user-defined experiments. This design goal is achieved by frequency mixing of an arbitrary waveform generator (AWG) output at the intermediate frequency (IF) with a constant source frequency (SF). Low noise SF at 250, 750, and 1000 MHz are available in the system. A wide range of frequencies from near-baseband to L-band can be generated as a result. Two-stage downconversion at the signal detection side is implemented that enables multi-frequency EPR capability. In the first stage, the signal frequency is converted to IF. A novel AWG-enabled digital auto-frequency control method that operates at IF is described that is used for automatic resonator tuning. Quadrature baseband EPR signal is generated in the second downconversion step. The semi-digital approach of mixing low-noise frequency sources with an AWG permits generation of arbitrary excitation patterns that include but are not limited to frequency sweeps for resonator tuning and matching, continuous-wave, and pulse sequences. Presented in this paper is the demonstration of rapid scan (RS) EPR imaging implemented at 800 MHz. Generation of stable magnetic scan waveforms is critical for the RS method. A digital automatic scan control (DASC) system was developed for sinusoidal magnetic field scans. DASC permits tight control of both amplitude and phase of the scans. A surface loop resonator was developed using 3D printing technology. RS EPR imaging system was validated using sample phantoms. In vivo imaging of a breast cancer mouse model is demonstrated.

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications.

Overhauser-enhanced Magnetic Resonance Imaging (OMRI) is a double resonance technique applied for oxygen imaging in aqueous samples and biological tissues. In this report, we present an improved OMRI approach of oxygen measurement using the single line “Finland” trityl spin probe. Compared to a traditional approach, we introduced an additional mechanism of leakage of spin polarization due to an interaction of a spin system with oxygen. The experimental comparison of the new approach with an oxygen-dependent leakage factor to a traditional approach performed in phantom samples in vitro, and mouse tumor model in vivo, shows improved accuracy of determination of oxygen and contrast agent concentrations.

Our previous study found that zinc finger protein 71 (ZNF71) mRNA expression was associated with chemosensitivity and its protein expression was prognostic of non-small-cell lung cancer (NSCLC). The Krüppel associated box (KRAB) transcriptional repression domain is commonly present in human zinc finger proteins, which are linked to imprinting, silencing of repetitive elements, proliferation, apoptosis, and cancer. This study revealed that ZNF71 KRAB had a significantly higher expression than the ZNF71 KRAB-less isoform in NSCLC tumors (n = 197) and cell lines (n = 117). Patients with higher ZNF71 KRAB expression had a significantly worse survival outcome than patients with lower ZNF71 KRAB expression (log-rank p = 0.04; hazard ratio (HR): 1.686 [1.026, 2.771]), whereas ZNF71 overall and KRAB-less expression levels were not prognostic in the same patient cohort. ZNF71 KRAB expression was associated with epithelial-to-mesenchymal transition (EMT) in both patient tumors and cell lines. ZNF71 KRAB was overexpressed in NSCLC cell lines resistant to docetaxel and paclitaxel treatment compared to chemo-sensitive cell lines, consistent with its association with poor prognosis in patients. Therefore, ZNF71 KRAB isoform is a more effective prognostic factor than ZNF71 overall and KRAB-less expression for NSCLC. Functional analysis using CRISPR-Cas9 and RNA interference (RNAi) screening data indicated that a knockdown/knockout of ZNF71 did not significantly affect NSCLC cell proliferation in vitro.

Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) has demonstrated notable clinical activity in cancer immunotherapy, but is limited by systemic toxicities, poor bioavailability, rapid clearance, and instability in vivo. Nanoparticles (NPs) may overcome these limitations and provide a mechanism for passive targeting of tumors. This study aimed to develop GM-CSF-loaded PLGA/PLGA-PEG NPs and evaluate them in vitro as a potential candidate for in vivo administration. NPs were created by a phase-separation technique that did not require toxic/protein-denaturing solvents or harsh agitation techniques and encapsulated GM-CSF in a more stable precipitated form. NP sizes were within 200 nm for enhanced permeability and retention (EPR) effect with negative zeta potentials, spherical morphology, and high entrapment efficiencies. The optimal formulation was identified by sustained release of approximately 70% of loaded GM-CSF over 24 hours, alongside an average size of 143 ± 35 nm and entrapment efficiency of 84 ± 5%. These NPs were successfully freeze-dried in 5% (w/v) hydroxypropyl-β-cyclodextrin for long-term storage and further characterized. Bioactivity of released GM-CSF was determined by observing GM-CSF receptor activation on murine monocytes and remained fully in-tact. NPs were not cytotoxic to murine bone marrow-derived macrophages (BMDMs) at concentrations up to 1 mg/mL as determined by MTT and Trypan Blue exclusion assays. Lastly, NP components generated no significant transcription of inflammation-regulating genes from BMDMs compared to IFNγ+LPS “M1” controls. This report lays the preliminary groundwork to validate in vivo studies with GM-CSF-loaded PLGA/PEG-PLGA NPs for tumor immunomodulation. Overall, these data suggest that in vivo delivery will be well tolerated.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1αfl/fl/LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2αfl/fl/LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α–deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α–deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α–deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical EPR due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert towards many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 ×10(8) M(−1)s(−1) leading to a tri-sulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔB(pp)=24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, P- nucleophiles opening access to various mono-functionalized sulfonated trityl radicals.

The comparison of fitness between cells leads to the elimination of less competent cells in the presence of more competent neighbors via cell competition (CC). This phenomenon has been linked with several cancer-related genes and thus may play an important role in cancer. Various processes are involved in the regulation of tumor initiation and growth, including tumor hypoxia, clonal stem cell selection, and immune cell response, all of which have been recently shown to have a potential connection with the mechanisms involved in CC. This review aims to unravel the relation between these processes and competitive cell interactions and how this affects disease progression.

Hypoxia leading to stabilization of hypoxia-inducible factor 1α (HIF-1α) serves as an early upstream initiator for adipose tissue (AT) dysfunction. Monocyte-derived macrophage infiltration in AT contributes to inflammation, fibrosis and obesity-related metabolic dysfunction. It was previously reported that myeloid cell-specific deletion of Hif-1α protected against high-fat diet (HFD)-induced AT dysfunction. Prolyl hydroxylases (PHDs) are key regulators of HIF-1α. We examined the effects of myeloid cell-specific upregulation and stabilization of Hif-1α via deletion of prolyl-hydroxylase 2 ( Phd2) and whether interleukin-1 receptor associated kinase-M ( Irak-M), a known downstream target of Hif-1α, contributes to Hif-1α-induced AT dysfunction. Our data show that with HFD, Hif-1α and Irak-M expressions were increased in the AT macrophages of Phd2flox/flox/ LysMcre mice compared with LysMcre mice. With HFD, Phd2flox/flox/ LysMcre mice exhibited increased AT inflammation, fibrosis, and systemic insulin resistance compared with control mice. Furthermore, Phd2flox/flox/ LysMcre mice bone marrow-derived macrophages exposed to hypoxia in vitro also had increased expressions of both Hif-1α and Irak-M. In wild-type mice, HFD induced upregulation of both HIF-1a and Irak-M in adipose tissue. Despite equivalent expression of Hif-1α compared with wild-type mice, globally-deficient Irak-M mice fed a HFD exhibited less macrophage infiltration, decreased inflammation and fibrosis and improved glucose tolerance. Global Irak-M deficiency was associated with an alternatively-activated macrophage phenotype in the AT after HFD. Together, these data show for the first time that an Irak-M-dependent mechanism likely mediates obesity-related AT dysfunction in conjunction with Hif-1α upregulation.

The etiology and pathogenesis of pulmonary fibrosis is poorly understood. We and others reported that M-CSF/CSF-1, M-CSF-R and downstream AKT activation plays an important role in lung fibrosis in mice models and in IPF patients. To understand potential molecular pathways used by M-CSF-R activation to direct lung fibrosis, we used a novel transgenic mouse model that expresses a constitutively-active form of AKT, myristoylated AKT (Myr-Akt), driven by the c-fms (M-CSF-R) promoter. We were particularly interested in the basal immune state of the lungs of these Myr-Akt mice to assess M-CSF-R-related priming for lung fibrosis. In support of a priming effect, macrophages isolated from the lungs of unchallenged Myr-Akt mice displayed an M2-tropism, enhanced co-expression of M-CSF-R and α-SMA, reduced autophagy reflected by reduced expression of the key autophagy genes Beclin-1, MAP1-Lc3a(Lc3a), and MAP1-Lc3b(Lc3b), and increased p62/STSQM1 expression compared with littermate WT mice. Furthermore, Myr-Akt mice had more basal circulating fibrocytes than WT mice. Lastly, upon bleomycin challenge, Myr-Akt mice showed enhanced collagen deposition, increased F4/80+ and CD45+ cells, reduced autophagy genes Beclin-1, Lc3a, and Lc3b expression, and a shorter life-span than WT littermates. These data provide support that M-CSF-R/AKT activation may have a priming effect which can predispose lung tissue to pulmonary fibrosis.

Postextraction alveolar bone loss, mostly affecting the buccal plate, occurs despite regenerative procedures. To better understand possible determinants, this prospective case series assesses gingival blood perfusion and tissue molecular responses in relation to postextraction regenerative outcomes.Adults scheduled to receive bone grafting in maxillary, non-molar, single-tooth extraction sites were recruited. Clinical documentation included the following: 1) probing depth (PD); 2) keratinized tissue width (KT); 3) tissue biotype (TB); and 4) plaque level. Wound closure was clinically evaluated. Gingival blood perfusion was measured by laser Doppler flowmetry (LDF). Wound fluid (WF) and gingival biopsies were analyzed for protein levels and gene expression, respectively, of relevant molecular markers. Bone healing outcomes were determined radiographically (cone-beam computed tomography). Healing was followed for 4 months.Data from 15 patients are reported. Postoperatively, neither complications nor changes in PD, KT, or TB were observed. LDF revealed decreased perfusion followed by hyperemia that persisted for 1 month (P ≤0.05). WF levels of angiopoietin-2, interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor peaked on day 6 (P ≤0.05) and decreased thereafter. Only IL-8 and TNF-α exhibited increased gene expression. Linear bone changes were negligible. Volumetric bone changes were minimal but statistically significant, with more bone loss when membrane was used (P = 0.05).Gingival blood perfusion after postextraction bone regenerative procedures follows an ischemia-reperfusion model. Transient increases in angiogenic factor levels and prolonged hyperemia characterize the soft tissue response. These soft tissue responses do not determine radiographic bone changes.

Extracellular vesicles (EVs) are mRNA-containing cell fragments shed into circulation during pathophysiological events. DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) regulate gene expression by modifying DNA methylation and altering transcription. Sepsis is a systemic insult resulting in vascular dysfunction, which can lead to shock and death. We analysed plasma from ICU patients for circulating EV numbers, defined as particles isolated from 1 mL plasma at 21,000xg, and DNMTs mRNA content as prognostic markers of septic shock. Compared to plasma from critically ill patients with or without sepsis, plasma from septic shock patients contained more EVs per mL, expressed as total DNMTs mRNAs over 5 days, and more individual DNMT mRNAs at each day. A comparison of EV-DNMT1 (maintenance methylation) with EV-DNMT3A+DNMT3B (de novo methylation) expression correlated highly with severity, and EVs from septic shock patients carried more total DNMT mRNAs and more DNMT3A+DNMT3B mRNAs than control or sepsis EVs. Total plasma EVs also correlated with sepsis severity. EV-DNMT mRNAs load, when coupled with total plasma EV number, may be a novel method to diagnose septic shock upon ICU admittance and offer opportunities to more precisely intervene with standard therapy or other targeted interventions to regulate EV release and/or specific DNMT activity.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

The purpose of this study is to investigate localized delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential therapeutic modality in breast cancer to alleviate tumor hypoxia and immunosuppression. A therapeutic that has the potential to target and reverse these factors of the tumor microenvironment (TME) would aid in reducing tumor aggressiveness, immune escape, metastasis, and importantly, could potentially be administered as a pre-treatment to reduce resistance to standard therapies such as chemotherapy and radiation. We have previously reported that 100 ng of intratumorally delivered recombinant GM-CSF 1) altered the macrophage profile from immunosuppressive M2-like to immunosupportive M1-like and 2) inhibited angiogenesis and induced near anoxic levels in the tumor due to the dose-dependent production of soluble VEGFR-1 from tumor macrophages that sequestered VEGF. Our current focus is to determine if a dosage window of GM-CSF exists that may normalize leaky and dysfunctional tumor vasculature and alleviate hypoxia in tumors by lesser sVEGFR-1 production, and simultaneously convert the immune cell profile from immunosuppressive to immunosupportive to promote cytotoxic T cell killing. After intratumoral injections with either GM-CSF or saline 3x per week for 2.5 weeks in an orthotopic PyMT murine breast cancer model, we utilized a soluble multifunctional trityl probe and Electron Paramagnetic Resonance Spectrometry to measure intratumoral oxygen and pH in vivo in real time. Our findings demonstrate that intratumoral injections of 15 ng GM-CSF rescued tumor oxygen by ~16% and raised pH ~0.2 units, while 5 ng enhanced oxygen even further by ~40% and raised pH ~0.4 units. Additionally, tumor-associated macrophages (TAMs) that were isolated from Stage IV MMTV-PyMT tumors and treated with 5 ng/ml GM-CSF 3x per week for 2 weeks demonstrated a reduction in IL-10 expression as determined by intracellular staining and flow cytometry. Further, as a result of these data, we have also formulated and characterized GM-CSF-containing PLGA/PEG-PLGA nanoparticles in vitro with average sizes under 200 nm for the purpose of systemic but localized delivery of dose-optimized recombinant GM-CSF to tumors. Nanoparticles yielded an entrapment efficiency of 87.9% +/- 4.2 S.D. and release of functional GM-CSF in vitro. Overall, we have demonstrated the potential of locally delivered recombinant GM-CSF to alleviate tumor hypoxia and acidity in vivo and reverse immunosuppressive TAM phenotype ex vivo, and we have formulated nanoparticles encapsulating recombinant murine GM-CSF as another modality which will be utilized for localized delivery of GM-CSF to breast tumors to achieve similar effects. Citation Format: Nicole Mihalik, Andrey Bobko, Valery Khramtsov, Benoit Driesschaert, Tim D. Eubank. Localized delivery of granulocyte-macrophage colony-stimulating factor to alleviate tumor hypoxia and immunosuppression in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2672.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO(2), pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO(2) and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction coefficients, and large Stokes shifts often above 100 nm. The probes are highly cell permeable, and exhibit stable bright fluorescence once intracellular; therefore are suited to the design of biosensors.

Psychological stressors are known to affect colonic diseases but the mechanisms by which this occurs, and whether probiotics can prevent stressor effects, are not understood. Because inflammatory monocytes that traffic into the colon can exacerbate colitis, we tested whether CCL2, a chemokine involved in monocyte recruitment, was necessary for stressor-induced exacerbation of infectious colitis. Mice were exposed to a social disruption stressor that entails repeated social defeat. During stressor exposure, mice were orally challenged with Citrobacter rodentium to induce a colonic inflammatory response. Exposure to the stressor during challenge resulted in significantly higher colonic pathogen levels, translocation to the spleen, increases in colonic macrophages, and increases in inflammatory cytokines and chemokines. The stressor-enhanced severity of C. rodentium-induced colitis was not evident in CCL2[superscript −/−] mice, indicating the effects of the stressor are CCL2-dependent. In addition, we tested whether probiotic intervention could attenuate stressor-enhanced infectious colitis by reducing monocyte/macrophage accumulation. Treating mice with probiotic Lactobacillus reuteri reduced CCL2 mRNA levels in the colon and attenuated stressor-enhanced infectious colitis. These data demonstrate that probiotic L. reuteri can prevent the exacerbating effects of stressor exposure on pathogen-induced colitis, and suggest that one mechanism by which this occurs is through downregulation of the chemokine CCL2., National Cancer Institute (U.S.) (Grants AT006552-01A1, P30-CA016058, and T32-DE014320)

Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO2), extracellular acidosis (pHe), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivo concurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO2, pHe and tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the “growth rate hypothesis” may underline its potential role in tumorigenesis and tumor progression.

Background Chronic inflammation is involved in the pathogenesis of human cervical spondylotic myelopathy and could also play a role in cervical spondylomyelopathy (CSM) in dogs. Hypothesis/Objectives That cerebrospinal fluid (CSF) cytokine concentrations would differ between clinically normal (control) and CSM-affected Great Danes (GDs), with affected GDs showing higher levels of inflammatory cytokines, such as interleukin (IL)-6 and monocyte chemoattractant protein-1/chemokine ligand 2 (MCP-1/CCL2). Animals Client-owned GDs: 15 control, 15 CSM-affected. Methods Prospective study. Dogs underwent cervical vertebral column magnetic resonance imaging and collection of CSF from the cerebellomedullary cistern. Cytokine concentrations were measured using a commercially available canine multiplex immunoassay. Cytokine concentrations were compared between groups. Associations with the administration of anti-inflammatory medications, disease duration and severity, severity of spinal cord (SC) compression, and SC signal changes were investigated in affected GDs. Results Affected GDs had significantly lower MCP-1/CCL2 (mean 138.03 pg/mL, 95% confidence interval [CI] = 114.85–161.20) than control GDs (212.89 pg/mL, 95% CI = 165.68–260.11, P = .028). In affected GDs, MCP-1/CCL2 concentrations correlated inversely with the severity of SC compression. There were no associations with administration of anti-inflammatory medications, disease duration, or disease severity. IL-6 concentrations were significantly higher (2.20 pg/mL, 95% CI = 1.92–2.47, P

Extracellular vesicles (EVs) are mRNA-containing cell fragments shed into circulation during pathophysiological events. DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) regulate gene expression by modifying DNA methylation and altering transcription. Sepsis is a systemic insult resulting in vascular dysfunction, which can lead to shock and death. We analysed plasma from ICU patients for circulating EV numbers, defined as particles isolated from 1 mL plasma at 21,000xg, and DNMTs mRNA content as prognostic markers of septic shock. Compared to plasma from critically ill patients with or without sepsis, plasma from septic shock patients contained more EVs per mL, expressed as total DNMTs mRNAs over 5 days, and more individual DNMT mRNAs at each day. A comparison of EV-DNMT1 (maintenance methylation) with EV-DNMT3A+DNMT3B (de novo methylation) expression correlated highly with severity, and EVs from septic shock patients carried more total DNMT mRNAs and more DNMT3A+DNMT3B mRNAs than control or sepsis EVs. Total plasma EVs also correlated with sepsis severity. EV-DNMT mRNAs load, when coupled with total plasma EV number, may be a novel method to diagnose septic shock upon ICU admittance and offer opportunities to more precisely intervene with standard therapy or other targeted interventions to regulate EV release and/or specific DNMT activity.

Reaction of peroxynitrite with coumarin boronic acid [1] was used for quantitation of peroxynitrite produced by activated murine macrophages (RAW 264.7) in cell culture. Highly fluorescent 7-hydroxycoumarin produced in reaction leads to fluorescence increase, affording real-time detection. Quiescent cells exhibit low rate of fluorescence increase which is further decreased in the presence of catalase, while catalase does not significantly affect the rate for activated cells. Specificity of the assay is supported by the dependence of the rate on the presence of arginine in the medium, inhibition by N6-(1-iminoethyl) L-lysine (inhibitor of iNOS) and by ebselen (peroxynitrite scavenger). The rate of peroxynitrite generation by stimulated RAW 264.7 was estimated as ~0.5 amol/(cell·s) that can be compared with NO generation rate of ~ 1.7 amol/(cell·s) determined earlier for the same cells [2]. Under the activation scheme used in this work (gamma-interferon + LPS), human THP-1 derived macrophages do not produce any peroxynitrite detectable by coumarin boronic acid. The level of iNOS mRNA in activated THP-1 derived macrophages does not exceed the level in control cells. 1 Zielonka, J.; Sikora, A.; Hardy, et al. Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chemical Research in Toxicology. (2012), 25 (9), 1793–1799. 2 Woldman, Y. Y., Eubank, T. D., Mock, A. J., et al. Detection of nitric oxide production in cell cultures by luciferine-luciferase chemiluminescence. Biochemical and Biophysical Research Communications (2015), 465 232-238