Retrograde AAV-mediated gene modulation reveals chloride intracellular channel proteins as potent regulators of retinal ganglion cell death.

Most projection neurons, including retinal ganglion cells (RGCs), undergo cell death after axotomy proximal to the cell body. Specific RGC subtypes, such as ON-OFF direction selective RGCs (ooDSGCs) are particularly vulnerable, whereas intrinsically photosensitive RGCs (ipRGCs) exhibit resilience to axonal injury. Through the application of RNA sequencing and fluorescent in situ hybridization, we show that the expression of chloride intracellular channel protein 1 and 4 (Clic1 and Clic4) are highly increased in the ooDSGCs after axonal injury. Toward determining a gene's role in RGCs, we optimized the utility and efficacy of adenovirus associated virus (AAV)-retro expressing short hairpin RNA (shRNA). Injection of AAV2-retro into the superior colliculus results in efficient shRNA expression in RGCs. Incorporating histone H2B gene fused with mGreenLantern results in bright nuclear reporter expression, thereby enhancing single RGC identification and cell quantitation in live retinas. Lastly, we demonstrate that AAV2-retro mediated knockdown of both Clic1 and Clic4 promotes RGC survival after injury. Our findings establish an integrated use of AAV2-retro-shRNA and real-time fundus imaging and reveal CLICs' contribution to RGC death.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

GSDMD deficiency ameliorates hyperoxia-induced BPD and ROP in neonatal mice.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are among the most common morbidities affecting extremely premature infants who receive oxygen therapy. Many clinical studies indicate that BPD is associated with advanced ROP. However, the mechanistic link between hyperoxia, BPD, and ROP remains to be explored. Gasdermin D (GSDMD) is a key executor of inflammasome-induced pyroptosis and inflammation. Inhibition of GSDMD has been shown to attenuate hyperoxia-induced BPD and brain injury in neonatal mice. The objective of this study was to further define the mechanistic roles of GSDMD in the pathogenesis of hyperoxia-induced BPD and ROP in mouse models. Here we show that global GSDMD knockout (GSDMD-KO) protects against hyperoxia-induced BPD by reducing macrophage infiltration, improving alveolarization and vascular development, and decreasing cell death. In addition, GSDMD deficiency prevented hyperoxia-induced ROP by reducing vasoobliteration and neovascularization, improving thinning of multiple retinal tissue layers, and decreasing microglial activation. RNA sequencing analyses of lungs and retinas showed that similar genes, including those from inflammatory, cell death, tissue remodeling, and tissue and vascular developmental signaling pathways, were induced by hyperoxia and impacted by GSDMD-KO in both models. These data highlight the importance of GSDMD in the pathogenesis of BPD and ROP and suggest that targeting GSDMD may be beneficial in preventing and treating BPD and ROP in premature infants.

Evaluation of Morlet Wavelet Analysis for Artifact Detection in Low-Frequency Commercial Near-Infrared Spectroscopy Systems.

Regional cerebral oxygen saturation (rSO2), a method of cerebral tissue oxygenation measurement, is recorded using non-invasive near-infrared Spectroscopy (NIRS) devices. A major limitation is that recorded signals often contain artifacts. Manually removing these artifacts is both resource and time consuming. The objective was to evaluate the applicability of using wavelet analysis as an automated method for simple signal loss artifact clearance of rSO2 signals obtained from commercially available devices. A retrospective observational study using existing populations (healthy control (HC), elective spinal surgery patients (SP), and traumatic brain injury patients (TBI)) was conducted. Arterial blood pressure (ABP) and rSO2 data were collected in all patients. Wavelet analysis was determined to be successful in removing simple signal loss artifacts using wavelet coefficients and coherence to detect signal loss artifacts in rSO2 signals. The removal success rates in HC, SP, and TBI populations were 100%, 99.8%, and 99.7%, respectively (though it had limited precision in determining the exact point in time). Thus, wavelet analysis may prove to be useful in a layered approach NIRS signal artifact tool utilizing higher-frequency data; however, future work is needed.

Immunotherapy, targeted therapy, and their cross talks in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a challenging malignancy with limited treatment options beyond surgery and chemotherapy. Recent advancements in targeted therapies and immunotherapy, including PD-1 and PD-L1 monoclonal antibodies, have shown promise, but their efficacy has not met expectations. Biomarker testing and personalized medicine based on genetic mutations and other biomarkers represent the future direction for HCC treatment. To address these challenges and opportunities, this comprehensive review discusses the progress made in targeted therapies and immunotherapies for HCC, focusing on dissecting the rationales, opportunities, and challenges for combining these modalities. The liver's unique physiology and the presence of fibrosis in many HCC patients pose additional challenges to drug delivery and efficacy. Ongoing efforts in biomarker development and combination therapy design, especially in the context of immunotherapies, hold promise for improving outcomes in advanced HCC. Through exploring the advancements in biomarkers and targeted therapies, this review provides insights into the challenges and opportunities in the field and proposes strategies for rational combination therapy design.

Targeting EZH2 to overcome the resistance to immunotherapy in lung cancer.

Unleashing the immune system to fight cancer has been a major breakthrough in cancer therapeutics since 2014 when anti-PD-1 antibodies (pembrolizumab and nivolumab) were approved for patients with metastatic melanoma. Therapeutic indications have rapidly expanded for many types of advanced cancer, including lung cancer. A variety of antibodies targeting the PD-1/PD-L1 checkpoint are contributing to this paradigm shift. The field now confronts two salient challenges: first, to improve the therapeutic outcome given the low response rate across the histologies; second, to identify biomarkers for improved patient selection. Pre-clinical and clinical studies are underway to evaluate combinatorial treatments to improve the therapeutic outcome paired with correlative studies to identify the factors associated with response and resistance. One of the emerging strategies is to combine epigenetic modifiers with immune checkpoint blockade (ICB) based on the evidence that targeting epigenetic elements can enhance anti-tumor immunity by reshaping the tumor microenvironment (TME). We will briefly review pleotropic biological functions of enhancer of zeste homolog 2 (EZH2), the enzymatic subunit of polycomb repressive complex 2 (PRC2), clinical developments of oral EZH2 inhibitors, and potentially promising approaches to combine EZH2 inhibitors and PD-1 blockade for patients with advanced solid tumors, focusing on lung cancer.

Evaluation of local tissue peri-implant reaction in total knee arthroplasty failure cases.

Introduction: Implant-related hypersensitivity is emerging as a causative factor as a potential source of total knee arthroplasty (TKA) failure. Mechanistically, this type IV hypersensitivity reaction (T4HR) is mediated by effector T-cells, macrophages, and leukocytes that infiltrate to the site of implant and react to metal exposure and induce inflammatory tissue damage. Methods: A case-control study was performed where cortical bone was taken at the time of revision surgery for all patients operated on for primary TKA in which metal allergy was suspected and for revision TKA cases done for presumed metal allergy. Cytof was used to determine the cell density of inflammatory cells, specifically Th1, Th2, M1, and M2 cells. Results: Comparing the mean cell density of primary versus revision TKA, revision TKA patients had significantly higher number of Th2 cells compared with Th1 cells (p = 0.0043). Among revision cases, there were significantly more M1 versus M2 macrophages (p = 0.034) within a patient. When comparing mean cell density of M1 versus M2 macrophages, there was a significant difference in both primary and revision TKA surgeries (p = 0.0041 primary, p < 0.001 revision). Among revision patients who had a predominance of Th2 cells, four (44%) of nine patients had a negative LTT/patch test. Conclusion: These data support metal hypersensitivity, mediated by a T4HR, for some cases of TKA failure. Current methods to screen patients for metal hypersensitivity prior to primary TKA have been inclusive. This study demonstrates the need for a more sensitive screening test from specimens in the knee joint, to more accurately identify patients who will exhibit a T4HR to metal.

Key Players of the Immunosuppressive Tumor Microenvironment and Emerging Therapeutic Strategies.

The tumor microenvironment (TME) is a complex, dynamic battlefield for both immune cells and tumor cells. The advent of the immune checkpoint inhibitors (ICI) since 2011, such as the anti-cytotoxic T-lymphocyte associated protein (CTLA)-4 and anti-programmed cell death receptor (PD)-(L)1 antibodies, provided powerful weapons in the arsenal of cancer treatments, demonstrating unprecedented durable responses for patients with many types of advanced cancers. However, the response rate is generally low across tumor types and a substantial number of patients develop acquired resistance. These primary or acquired resistance are attributed to various immunosuppressive elements (soluble and cellular factors) and alternative immune checkpoints in the TME. Therefore, a better understanding of the TME is absolutely essential to develop therapeutic strategies to overcome resistance. Numerous clinical studies are underway using ICIs and additional agents that are tailored to the characteristics of the tumor or the TME. Some of the combination treatments are already approved by the Food and Drug Administration (FDA), such as platinum-doublet chemotherapy, tyrosine kinase inhibitor (TKI) -targeting vascular endothelial growth factor (VEGF) combined with anti-PD-(L)1 antibodies or immuno-immuno combinations (anti-CTLA-4 and anti-PD-1). In this review, we will discuss the key immunosuppressive cells, metabolites, cytokines or chemokines, and hypoxic conditions in the TME that contribute to tumor immune escape and the prospect of relevant clinical trials by targeting these elements in combination with ICIs.

Transfer of hepatocellular microRNA regulates cytochrome P450 2E1 in renal tubular cells.

BACKGROUND: Extracellular microRNAs enter kidney cells and modify gene expression. We used a Dicer-hepatocyte-specific microRNA conditional-knock-out (Dicer-CKO) mouse to investigate microRNA transfer from liver to kidney. METHODS: Dicerflox/flox mice were treated with a Cre recombinase-expressing adenovirus (AAV8) to selectively inhibit hepatocyte microRNA production (Dicer-CKO). Organ microRNA expression was measured in health and following paracetamol toxicity. The functional consequence of hepatic microRNA transfer was determined by measuring the expression and activity of cytochrome P450 2E1 (target of the hepatocellular miR-122), and by measuring the effect of serum extracellular vesicles (ECVs) on proximal tubular cell injury. In humans with liver injury we measured microRNA expression in urinary ECVs. A murine model of myocardial infarction was used as a non-hepatic model of microRNA release. FINDINGS: Dicer-CKO mice demonstrated a decrease in kidney miR-122 in the absence of other microRNA changes. During hepatotoxicity, miR-122 increased in kidney tubular cells; this was abolished in Dicer-CKO mice. Depletion of hepatocyte microRNA increased kidney cytochrome P450 2E1 expression and activity. Serum ECVs from mice with hepatotoxicity increased proximal tubular cell miR-122 and prevented cisplatin toxicity. miR-122 increased in urinary ECVs during human hepatotoxicity. Transfer of microRNA was not restricted to liver injury -miR-499 was released following cardiac injury and correlated with an increase in the kidney. INTERPRETATION: Physiological transfer of functional microRNA to the kidney is increased by liver injury and this signalling represents a new paradigm for understanding the relationship between liver injury and renal function. FUNDING: Kidney Research UK, Medical Research Scotland, Medical Research Council.

Definition of Haptens Derived from Sulfamethoxazole: In Vitro and in Vivo.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.

p35 hemizygosity activates Akt but does not improve motor function in the YAC128 mouse model of Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder resulting from N-terminal polyglutamine expansion in the huntingtin protein. A relatively selective and early loss of medium spiny neurons in the striatum is a hallmark of HD neuropathology. Although the exact mechanism of mutant huntingtin-mediated neurodegeneration is unclear, recent evidence suggests that NMDA-receptor-mediated excitotoxicity is involved. Our previously published findings show that decreasing levels of the cdk5 activators, p35 and p25, reduces NMDA receptor-mediated excitotoxicity in striatal neurons in vivo. In this study we directly examined the effect of reducing levels of p35 and p25 in the context of mutant huntingtin toxicity, using the B6 YAC128 mouse model of HD. Our findings demonstrate that deletion of a single allele of p35 in the B6 YAC128 mice results in an upregulation of Akt activity, and increases phosphorylation of mutant huntingtin at Ser421. Longitudinal behavioral analysis showed that this 50% reduction in p35 and p25 levels did not improve accelerating Rotarod performance in these YAC128 mice. However, a complete deletion of p35 normalized the accelerating Rotarod performance relative to their non-transgenic littermates at four months of age.

Regenerative Responses and Axon Pathfinding of Retinal Ganglion Cells in Chronically Injured Mice.

Purpose: Enhanced regeneration of retinal ganglion cell (RGC) axons can be achieved by modification of numerous neuronal-intrinsic factors. However, axon growth initiation and the pathfinding behavior of these axons after traumatic injury remain poorly understood outside of acute injury paradigms, despite the clinical relevance of more chronic settings. We therefore examined RGC axon regeneration following therapeutic delivery that is postponed until 2 months after optic nerve crush injury. Methods: Optic nerve regeneration was induced by virally mediated (adeno-associated virus) ciliary neurotrophic factor (AAV-CNTF) administered either immediately or 56 days after optic nerve crush in wild-type or Bax knockout (KO) mice. Retinal ganglion nerve axon regeneration was assessed 21 and 56 days after viral injection. Immunohistochemical analysis of RGC injury signals and extrinsic factors in the optic nerve were also examined at 5 and 56 days post crush. Results: In addition to sustained expression of injury response proteins in surviving RGCs, we observe axon regrowth in wild-type and apoptosis-deficient Bax KO mice following AAV-CNTF treatment. Fewer instances of aberrant axon growth are seen, at least in the area near the lesion site, in animals given treatment 56 days after crush injury compared to the animals given treatment immediately after injury. We also find evidence of long distance growth into a visual target in Bax KO mice despite postponed initiation of this regenerative program. Conclusions: These studies provide evidence against an intrinsic critical period for RGC axon regeneration or degradation of injury signals. Regeneration results from Bax KO mice imply highly sustained regenerative capacity in RGCs, highlighting the importance of long-lasting neuroprotective strategies as well as of RGC axon guidance research in chronically injured animals.

Proteomic analysis of pancreatic cancer stem cells: Functional role of fatty acid synthesis and mevalonate pathways.

Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated. To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins. Pathway analysis revealed activation of glycolysis, the pentose phosphate pathway, the pyruvate-malate cycle, and lipid metabolism as well as downregulation of the Krebs cycle, the splicesome and non-homologous end joining. These findings were supported by metabolomics and immunoblotting analysis. It was also found that inhibition of fatty acid synthase by cerulenin and of mevalonate pathways by atorvastatin have a greater anti-proliferative effect on cancer stem cells than parental cells. Taken together, these results clarify some important aspects of the metabolic network signature of pancreatic cancer stem cells, shedding light on key and novel therapeutic targets and suggesting that fatty acid synthesis and mevalonate pathways play a key role in ensuring their viability. BIOLOGICAL SIGNIFICANCE: To better understand the altered metabolic pathways of pancreatic cancer stem cells (CSCs), a comprehensive proteomic analysis and metabolite profiling investigation of Panc1 and Panc1 CSCs were carried out. The findings obtained indicate that Panc1 CSCs are characterized by upregulation of glycolysis, pentose phosphate pathway, pyruvate-malate cycle, and lipid metabolism and by downregulation of Krebs cycle, spliceosome and non-homologous end joining. Moreover, fatty acid synthesis and mevalonate pathways are shown to play a critical contribution to the survival of pancreatic cancer stem cells. This study is helpful for broadening the knowledge of pancreatic cancer stem cells and could accelerate the development of novel therapeutic strategies.

Return to Golf After Lumbar Fusion.

BACKGROUND: Spinal fusion surgery is being increasingly performed, yet few studies have focused on return to recreational sports after lumbar fusion and none have specifically analyzed return to golf. HYPOTHESIS: Most golfers successfully return to sport after lumbar fusion surgery. STUDY DESIGN: Case series. LEVEL OF EVIDENCE: Level 4. METHODS: All patients who underwent 1- or 2-level primary lumbar fusion surgery for degenerative pathologies performed by a single surgeon between January 2008 and October 2012 and had at least 1-year follow-up were included. Patients completed a specifically designed golf survey. Surveys were mailed, given during follow-up clinic, or answered during telephone contact. RESULTS: A total of 353 patients met the inclusion and exclusion criteria, with 200 responses (57%) to the questionnaire producing 34 golfers. The average age of golfers was 57 years (range, 32-79 years). In 79% of golfers, preoperative back and/or leg pain significantly affected their ability to play golf. Within 1 year from surgery, 65% of patients returned to practice and 52% returned to course play. Only 29% of patients stated that continued back/leg pain limited their play. Twenty-five patients (77%) were able to play the same amount of golf or more than before fusion surgery. Of those providing handicaps, 12 (80%) reported the same or an improved handicap. CONCLUSION: More than 50% of golfers return to on-course play within 1 year of lumbar fusion surgery. The majority of golfers can return to preoperative levels in terms of performance (handicap) and frequency of play. CLINICAL RELEVANCE: This investigation offers insight into when golfers return to sport after lumbar fusion surgery and provides surgeons with information to set realistic expectations postoperatively.

Virally delivered, constitutively active NFκB improves survival of injured retinal ganglion cells.

As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno-associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2-p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P-value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P-value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration.

Enhanced Transcriptional Activity and Mitochondrial Localization of STAT3 Co-induce Axon Regrowth in the Adult Central Nervous System.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor central to axon regrowth with an enigmatic ability to act in different subcellular regions independently of its transcriptional roles. However, its roles in mature CNS neurons remain unclear. Here, we show that along with nuclear translocation, STAT3 translocates to mitochondria in mature CNS neurons upon cytokine stimulation. Loss- and gain-of-function studies using knockout mice and viral expression of various STAT3 mutants demonstrate that STAT3's transcriptional function is indispensable for CNS axon regrowth, whereas mitochondrial STAT3 enhances bioenergetics and further potentiates regrowth. STAT3's localization, functions, and growth-promoting effects are regulated by mitogen-activated protein kinase kinase (MEK), an effect further enhanced by Pten deletion, leading to extensive axon regrowth in the mouse optic pathway and spinal cord. These results highlight CNS neuronal dependence on STAT3 transcriptional activity, with mitochondrial STAT3 providing ancillary roles, and illustrate a critical contribution for MEK in enhancing diverse STAT3 functions and axon regrowth.

Secretome protein signature of human pancreatic cancer stem-like cells.

Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9. Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified. BIOLOGICAL SIGNIFICANCE: The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-1 CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9. We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered.

Factors associated with failure of nonoperative treatment in lateral epicondylitis.

BACKGROUND: Lateral epicondylitis is a common cause of elbow pain that is treated with a variety of nonoperative measures and often improves with time. Minimal research is available on patients in whom these nonoperative treatments fail. PURPOSE: To identify baseline patient and disease factors associated with the failure of nonoperative treatment of lateral epicondylitis, defined as surgery after a period of nonoperative treatment. STUDY DESIGN: Case control study; Level of evidence, 3. METHODS: A total of 580 patients treated for lateral epicondylitis at a tertiary center between 2007 and 2012 were analyzed. Disease-specific and patient demographic characteristics were compared between patient groups (nonoperative vs surgical treatment). A multivariable logistic regression model was created based on preliminary univariate testing to determine which characteristics were associated with failure of nonoperative treatment. RESULTS: Of the 580 patients, 92 (16%) underwent surgical treatment at a mean of 6 months (range, 0-31 months) from their initial visit. Univariate analysis demonstrated a potential association (P < .10) between operative management and the following factors at initial diagnosis: increased age, body mass index, duration of symptoms, presence of radial tunnel syndrome, prior injection, physical therapy, splinting, smoking, workers' compensation, a labor occupation, use of narcotics, use of antidepressant medications, and previous orthopaedic surgery. In the final multivariable model, a workers' compensation claim (odds ratio [OR], 8.1), prior injection (OR, 5.6), the presence of radial tunnel syndrome (OR, 3.1), previous orthopaedic surgery (OR, 3.2), and duration of symptoms >12 months (OR, 2.5) remained significant independent predictors of surgical treatment. CONCLUSION: This study identifies risk factors for surgical treatment for lateral epicondylitis. While these findings do not provide information regarding causal factors associated with surgery, these patient and disease-specific considerations may be helpful when counseling patients regarding treatment options and the likelihood of the success of continued nonoperative treatment.

3D Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates

The histological assessment of spinal cord tissue in three dimensions has previously been very time consuming and prone to errors of interpretation. Advances in tissue clearing have significantly improved visualization of fluorescently labelled axons. While recent proof-of-concept studies have been performed with transgenic mice in which axons were prelabeled with GFP, investigating axonal regeneration requires stringent axonal tracing methods as well as the use of animal models in which transgenic axonal labeling is not available. Using rodent models of spinal cord injury, we labeled axon tracts of interest using both adeno-associated virus and chemical tracers and performed tetrahydrofuran-based tissue clearing to image multiple axon types in spinal cords using light sheet and confocal microscopy. Using this approach, we investigated the relationships between axons and scar-forming cells at the injury site as well as connections between sensory axons and motor pools in the spinal cord. In addition, we used these methods to trace axons in nonhuman primates. This reproducible and adaptable virus-based approach can be combined with transgenic mice or with chemical-based tract-tracing methods, providing scientists with flexibility in obtaining axonal trajectory information from transparent tissue.

Total proteome analysis identifies migration defects as a major pathogenetic factor in immunoglobulin heavy chain variable region (IGHV)-unmutated chronic lymphocytic leukemia.

The mutational status of the immunoglobulin heavy chain variable region defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and unmutated (UM-CLL). To elucidate the molecular mechanisms underlying the adverse clinical outcome associated with UM-CLL, total proteomes from nine UM-CLL and nine M-CLL samples were analyzed by isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry. Based on the expression of 3521 identified proteins, principal component analysis separated CLL samples into two groups corresponding to immunoglobulin heavy chain variable region mutational status. Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells underexpressed proteins associated with cytoskeletal remodeling and overexpressed proteins associated with transcriptional and translational activity. Taken together, our findings indicate that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes, where they are exposed to proliferative stimuli. In keeping with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed a strong and specific association between UM-CLL and lymphadenopathy. Our study illustrates the potential of total proteome analysis to elucidate pathogenetic mechanisms in cancer.