Optimization of a mouse model of pancreatic cancer to simulate the human phenotypes of metastasis and cachexia.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) presents with a high mortality rate. Two important features of PDAC contribute to this poor outcome. The first is metastasis which occurs in ~ 80% of PDAC patients. The second is cachexia, which compromises treatment tolerance for patients and reduces their quality of life. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. METHODS: Here, we optimize an orthotopic mouse model of PDAC by altering several conditions, including the subcloning of parental murine PDAC cells, implantation site, number of transplanted cells, and age of recipient mice. We perform spatial profiling to compare primary and metastatic immune microenvironments and RNA sequencing to gain insight into the mechanisms of muscle wasting in PDAC-induced cachexia, comparing non-metastatic to metastatic conditions. RESULTS: These modifications extend the time course of the disease and concurrently increase the rate of metastasis to approximately 70%. Furthermore, reliable cachexia endpoints are achieved in both PDAC mice with and without metastases, which is reminiscent of patients. We also find that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. CONCLUSION: Together, this model is likely to be advantageous in both advancing our understanding of the mechanism of PDAC cachexia, as well as in the evaluation of novel therapeutics.

Cancer cachexia: involvement of an expanding macroenvironment.

Advanced cancers often present with the cachexia syndrome that impacts peripheral tissues, leading to involuntary weight loss and reduced prognosis. The central tissues undergoing depletion are skeletal muscle and adipose, but recent findings reveal an expanding tumor macroenvironment involving organ crosstalks that underlie the cachectic state.

Targeting protein tyrosine phosphatases for CDK6-induced immunotherapy resistance.

Elucidating the mechanisms of resistance to immunotherapy and developing strategies to improve its efficacy are challenging goals. Bioinformatics analysis demonstrates that high CDK6 expression in melanoma is associated with poor progression-free survival of patients receiving single-agent immunotherapy. Depletion of CDK6 or cyclin D3 (but not of CDK4, cyclin D1, or D2) in cells of the tumor microenvironment inhibits tumor growth. CDK6 depletion reshapes the tumor immune microenvironment, and the host anti-tumor effect depends on cyclin D3/CDK6-expressing CD8+ and CD4+ T cells. This occurs by CDK6 phosphorylating and increasing the activities of PTP1B and T cell protein tyrosine phosphatase (TCPTP), which, in turn, decreases tyrosine phosphorylation of CD3ζ, reducing the signal transduction for T cell activation. Administration of a PTP1B and TCPTP inhibitor prove more efficacious than using a CDK6 degrader in enhancing T cell-mediated immunotherapy. Targeting protein tyrosine phosphatases (PTPs) might be an effective strategy for cancer patients who resist immunotherapy treatment.

Impact of intraoperative methadone use on postoperative opioid requirements after cardiac surgery.

PURPOSE: Inadequate pain control after cardiac surgery increases postoperative morbidity. Increasing evidence suggests that perioperative intravenous (IV) methadone results in improved analgesia. This study evaluated the effect of intraoperative IV methadone on postoperative opioid requirements and surgical recovery. METHODS: A retrospective review of patients undergoing coronary artery bypass graft (CABG), valvular surgery or both between April 2017 and August 2018 was conducted. Patients were separated into a usual care cohort of those who received short-acting opioids (ie, IV fentanyl, hydromorphone, or morphine) alone or a methadone cohort of those who received IV methadone plus short-acting opioids. Opioid requirements were assessed within the first 24 hours of surgery (postoperative day [POD] 0) and 25 to 48 hours after surgery (POD 1) as oral morphine milligram equivalents (MME). Postoperative pain scores, adjunctive analgesia, time to extubation, use of noninvasive respiratory support (continuous positive airway pressure [CPAP] or bilevel positive airway pressure [BiPAP]), and intensive care unit (ICU) and hospital length of stay (LOS) were also evaluated. RESULTS: A total of 117 patients were evaluated (methadone cohort, n = 52; usual care cohort, n = 65). Median cumulative intraoperative opioid consumption was less in the methadone cohort (150 MME vs 314.1 MME; P < 0.0001). The methadone cohort required 44% fewer MME than the usual care cohort on POD 0 (median MME, 15.8 vs 36; P = 0.025), with low and not significantly different opioid use in both cohorts on POD 1 (15.5 MME vs 7.5 MME; P = 0.47). Weight-based methadone dosing ranged from 0.1 to 0.4 mg/kg (mean, 0.22 mg/kg). There were no significant differences in pain scores, time to extubation, use of CPAP or BiPAP, or ICU and hospital LOS. CONCLUSION: Intraoperative IV methadone in cardiac surgery patients was safe and significantly reduced intraoperative and postoperative opioid requirements on POD 0.

Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth.

The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3ß (GSKß), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.

NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.

NF-κB regulates GDF-15 to suppress macrophage surveillance during early tumor development.

Macrophages are attracted to developing tumors and can participate in immune surveillance to eliminate neoplastic cells. In response, neoplastic cells utilize NF-κB to suppress this killing activity, but the mechanisms underlying their self-protection remain unclear. Here, we report that this dynamic interaction between tumor cells and macrophages is integrally linked by a soluble factor identified as growth and differentiation factor 15 (GDF-15). In vitro, tumor-derived GDF-15 signals in macrophages to suppress their proapoptotic activity by inhibiting TNF and nitric oxide (NO) production. In vivo, depletion of GDF-15 in Ras-driven tumor xenografts and in an orthotopic model of pancreatic cancer delayed tumor development. This delay correlated with increased infiltrating antitumor macrophages. Further, production of GDF-15 is directly regulated by NF-κB, and the colocalization of activated NF-κB and GDF-15 in epithelial ducts of human pancreatic adenocarcinoma supports the importance of this observation. Mechanistically, we found that GDF-15 suppresses macrophage activity by inhibiting TGF-ß-activated kinase (TAK1) signaling to NF-κB, thereby blocking synthesis of TNF and NO. Based on these results, we propose that the NF-κB/GDF-15 regulatory axis is important for tumor cells in evading macrophage immune surveillance during the early stages of tumorigenesis.

Enhancing the Cell Permeability and Metabolic Stability of Peptidyl Drugs by Reversible Bicyclization.

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. A general, reversible bicyclization strategy is now reported to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell-penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell-permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was applied to generate a cell-permeable bicyclic peptidyl inhibitor against the NEMO-IKK interaction.

A novel approach to symptomatic lumbar facet joint synovial cyst injection and rupture using iGuide navigational software: A case report and review.

Spinal synovial cysts are benign protrusions of facet joint capsules caused by degenerative spondylosis, most frequently involving the L4-5 level, and commonly lead to symptoms of back pain, radiculopathy and neurogenic claudication. Although percutaneous treatment via facet joint steroid injection with cyst rupture can provide significant symptom relief, cyst rupture is not always achievable via an indirect trans-facet approach due to limited access from severe degenerative changes. In this case, we describe a successful approach to direct cyst access using a laser-guided navigational software in a patient with severe facet joint osteophytosis. We provide a brief review of literature.

An NF-κB--EphrinA5-Dependent Communication between NG2(+) Interstitial Cells and Myoblasts Promotes Muscle Growth in Neonates.

Skeletal muscle growth immediately following birth is critical for proper body posture and locomotion. However, compared with embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight into this process by revealing a unique NF-κB-dependent communication between NG2(+) interstitial cells and myoblasts. NF-κB in NG2(+) cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2(+) cells, which we further deduce is an NF-κB target gene. Together, these results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth.

NF-κB functions in tumor initiation by suppressing the surveillance of both innate and adaptive immune cells.

NF-κB is considered a major contributor to tumor development, but how this factor functions in the initial stages of oncogenesis is not clear. In a model of Ras-induced transformation, we probed NF-κB function as preneoplastic cells formed tumors in mice. As previously shown, the p65 subunit of NF-κB acts as a tumor suppressor in normal cells by sustaining senescence following DNA damage. Our current data reveal that, following immortalization, p65 switches to an oncogene by counteracting the surveillance properties of immune cells. NF-κB exerts this effect by protecting transformed cells against macrophage-derived proapoptotic factors, tumor necrosis factor, and nitric oxide. Additionally, NF-κB acts through transforming growth factor beta (TGF-ß) to mitigate T cell cytotoxicity and other factors to expand myeloid-derived suppressor cells. Together, these data suggest that NF-κB functions in the early stages of transformation by suppressing immune surveillance of both innate and adaptive immune cells, information that may be useful for targeted immunotherapies.

Child implant users' imitation of happy- and sad-sounding speech.

Cochlear implants have enabled many congenitally or prelingually deaf children to acquire their native language and communicate successfully on the basis of electrical rather than acoustic input. Nevertheless, degraded spectral input provided by the device reduces the ability to perceive emotion in speech. We compared the vocal imitations of 5- to 7-year-old deaf children who were highly successful bilateral implant users with those of a control sample of children who had normal hearing. First, the children imitated several happy and sad sentences produced by a child model. When adults in Experiment 1 rated the similarity of imitated to model utterances, ratings were significantly higher for the hearing children. Both hearing and deaf children produced poorer imitations of happy than sad utterances because of difficulty matching the greater pitch modulation of the happy versions. When adults in Experiment 2 rated electronically filtered versions of the utterances, which obscured the verbal content, ratings of happy and sad utterances were significantly differentiated for deaf as well as hearing children. The ratings of deaf children, however, were significantly less differentiated. Although deaf children's utterances exhibited culturally typical pitch modulation, their pitch modulation was reduced relative to that of hearing children. One practical implication is that therapeutic interventions for deaf children could expand their focus on suprasegmental aspects of speech perception and production, especially intonation patterns.

Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity.

Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition-mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition-induced cytotoxicity.

Regulation of the let-7a-3 promoter by NF-κB.

Changes in microRNA expression have been linked to a wide array of pathological states. However, little is known about the regulation of microRNA expression. The let-7 microRNA is a tumor suppressor that inhibits cellular proliferation and promotes differentiation, and is frequently lost in tumors. We investigated the transcriptional regulation of two let-7 family members, let-7a-3 and let-7b, which form a microRNA cluster and are located 864 bp apart on chromosome 22q13.31. Previous reports present conflicting data on the role of the NF-κB transcription factor in regulating let-7. We cloned three fragments upstream of the let-7a-3/let-7b miRNA genomic region into a plasmid containing a luciferase reporter gene. Ectopic expression of subunits of NF-κB (p50 or p65/RelA) significantly increased luciferase activity in HeLa, 293, 293T and 3T3 cells, indicating that the let-7a-3/let-7b promoter is highly responsive to NF-κB. Mutation of a putative NF-κB binding site at bp -833 reduced basal promoter activity and decreased promoter activity in the presence of p50 or p65 overexpression. Mutation of a second putative binding site, at bp -947 also decreased promoter activity basally and in response to p65 induction, indicating that both sites contribute to NF-κB responsiveness. While the levels of the endogenous primary let-7a and let-7b transcript were induced in response to NF-κB overexpression in 293T cells, the levels of fully processed, mature let-7a and let-7b miRNAs did not increase. Instead, levels of Lin-28B, a protein that blocks let-7 maturation, were induced by NF-κB. Increased Lin-28B levels could contribute to the lack of an increase in mature let-7a and let-7b. Our results suggest that the final biological outcome of NF-κB activation on let-7 expression may vary depending upon the cellular context. We discuss our results in the context of NF-κB activity in repressing self-renewal and promoting differentiation.

No evidence of a link between influenza vaccines and Guillain-Barre syndrome-associated antiganglioside antibodies.

BACKGROUND: Guillain-Barre syndrome (GBS) is a rare autoimmune disease characterized by acute, progressive peripheral neuropathy and is commonly associated with the presence of antiganglioside antibodies. Previously, influenza vaccination was linked with the increased incidence of GBS; however, whether antiganglioside antibodies are subsequently induced remains unresolved. METHODS: Sera from human subjects vaccinated with seasonal influenza vaccines from the 2007-2008, 2008-2009, or 1976-1977 influenza seasons were screened for the induction of immunity to influenza and the presence of antiganglioside antibodies pre- and post-vaccination. Likewise, sera from mice vaccinated with seasonal influenza vaccines (1988-1989, 2007-2008) or "swine flu" pandemic vaccines (1976, 2009) were assessed in the same manner. Viruses were also screened for cross-reacting ganglioside epitopes. RESULTS: Antiganglioside antibodies were found to recognize influenza viruses; this reactivity correlated with virus glycosylation. Antibodies to influenza viruses were detected in human and mouse sera, but the prevalence of antiganglioside antibodies was extremely low. CONCLUSIONS: Although the correlation between antiganglioside antibody cross-reactivity and glycosylation of viruses suggests the role of shared carbohydrate epitopes, no correlation was observed between hemagglutinin-inhibition titers and the induction of antiganglioside antibodies after influenza vaccination.

A novel platform for biologically active recombinant human interleukin-13 production.

Interleukin-13 (IL-13) is a pleiotropic regulatory cytokine with the potential for treating several human diseases, including type-1 diabetes. Thus far, conventional expression systems for recombinant IL-13 production have proven difficult and are limited by efficiency. In this study, transgenic plants were used as a novel expression platform for the production of human IL-13 (hIL-13). DNA constructs containing hIL-13 cDNA were introduced into tobacco plants. Transcriptional expression of the hIL-13 gene in transgenic plants was confirmed by reverse transcriptase-polymerase chain reaction and Northern blotting. Western blot analysis showed that the hIL-13 protein was efficiently accumulated in transgenic plants and present in multiple molecular forms, with an expression level as high as 0.15% of total soluble protein in leaves. The multiple forms of plant-derived recombinant hIL-13 (rhIL-13) are a result of differential N-linked glycosylation, as revealed by enzymatic and chemical deglycosylation, but not of disulphide-linked oligomerization. In vitro trypsin digestion indicated that plant rhIL-13 was more resistant than unglycosylated control rhIL-13 to proteolysis. The stability of plant rhIL-13 to digestion was further supported with simulated gastric and intestinal fluid digestion. In vitro bioassays using a factor-dependent human erythroleukaemic cell line (TF-1 cells) showed that plant rhIL-13 retained the biological functions of the authentic hIL-13 protein. These results demonstrate that transgenic plants are superior to conventional cell-based expression systems for the production of rhIL-13. Moreover, transgenic plants synthesizing high levels of rhIL-13 may prove to be an attractive delivery system for direct oral administration of IL-13 in the treatment of clinical diseases such as type-1 diabetes.

Diuretics: still the mainstay of treatment.

The mainstay of treatment of acute decompensated heart failure is diuretic therapy. While there are no data showing a morbidity or mortality benefit from the use of chronic diuretic therapy, diuretics rapidly improve symptoms associated with volume overload. Thus, despite concerns that some diuretics may cause harm by neurohormonal activation, these agents continue to be the first-line treatment for patients with heart failure. There is no conclusive evidence that one means of diuresis is better than another. When administration of moderate doses of loop diuretics is not sufficient, patients can be treated with higher doses, continuous infusions, or the addition of a thiazide diuretic or aldosterone antagonist. Diuretics improve symptoms but should be used in addition to other agents that improve the long-term outcome of patients with heart failure.

The characteristics and prognostic importance of NT-ProBNP concentrations in critically ill patients.

BACKGROUND: There are limited data for prognostic and diagnostic use of natriuretic peptides in intensive care unit (ICU) patients. We evaluate amino-terminal brain natriuretic peptide (NT-proBNP) in the medical ICU, specifically its correlation with noncardiac admission diagnosis and prognosis of critically ill patients. METHODS: NT-proBNP (pg/mL) was measured in 179 ICU patients without acute decompensated heart failure or acute coronary syndrome. Death during hospitalization (mortality), APACHE II score, laboratory data, echocardiograms, medical history, and demographics were assessed. NT-proBNP concentrations were compared with established cutoffs for congestive heart failure (>450 pg/mL for patients <50 years of age, >900 pg/mL for patients 50-70 years of age, and >1800 pg/mL for patients >70 years of age). Predictors of mortality and of NT-proBNP were analyzed by regression analysis. Tertiles were compared by analysis of variance and chi-squared test. RESULTS: NT-proBNP was elevated in these ICU patients (median 2139 pg/mL, 25th percentile 540 pg/mL, 75% percentile 7389 pg/mL). Severity of illness and renal dysfunction (APACHE II score and serum creatinine) increased with rising NT-proBNP. The incidence of acute respiratory distress syndrome, sepsis, death, history of coronary artery disease (CAD) or congestive heart failure (all P <.05) increased with each tertile. Independent predictors of increased NT-proBNP were creatinine (P <.001), CAD (P <.001), APACHE II score (P <.05), and sepsis (P < or =.001). Overall hospital mortality was 26%, and log NT-proBNP (P <.05), APACHE II (P < or =.001), and CAD (P <.05) were independent predictors of mortality. CONCLUSIONS: For patients admitted to the ICU without decompensated heart failure or acute coronary syndrome, NT-proBNP concentrations are markedly elevated, especially in patients with sepsis. NT-proBNP strongly and independently predicts mortality. However, NT-proBNP should not be used to direct volume management in critically ill patients.

Impedance cardiography: more questions than answers.

Thoracic electrical bioimpedance, also known as impedance cardiography (ICG), is a noninvasive method to obtain hemodynamic measurements, including cardiac output. Recently, there has been a flurry of reports on the clinical use of ICG. Authors have suggested that ICG measurements are useful for a myriad of situations, including diagnosis of heart failure, monitoring of a patient's clinical status, and assisting in medicine titration decisions. However, data continue to suggest poor correlation between current generation ICG devices and invasive measurements of cardiac output, especially in heart failure patients. ICG is also not able to accurately measure left ventricular filling pressures. There are limited data demonstrating any improved outcomes using ICG in the clinical setting. Given the available data, ICG use should be limited to the research setting.

Impedance cardiography: more questions than answers.

Thoracic electrical bioimpedance, also known as impedance cardiography (ICG), is a noninvasive method to obtain hemodynamic measurements, including cardiac output. Recently, there has been a flurry of reports on the clinical use of ICG. Authors have suggested that ICG measurements are useful for a myriad of situations, including diagnosis of heart failure, monitoring of a patient's clinical status, and assisting in medicine titration decisions. However, data continue to suggest poor correlation between current generation ICG devices and invasive measurements of cardiac output, especially in heart failure patients. ICG is also not able to accurately measure left ventricular filling pressures. There are limited data demonstrating any improved outcomes using ICG in the clinical setting. Given the available data, ICG use should be limited to the research setting.