Age-associated changes in electroretinography measures in companion dogs.

PURPOSE: To determine the association between age and retinal full-field electroretinographic (ERG) measures in companion (pet) dogs, an important translational model species for human neurologic aging. METHODS: Healthy adult dogs with no significant ophthalmic abnormalities were included. Unilateral full-field light- and dark-adapted electroretinography was performed using a handheld device, with mydriasis and topical anaesthesia. Partial least squares effect screening analysis was performed to determine the effect of age, sex, body weight and use of anxiolytic medication on log-transformed ERG peak times and amplitudes; age and anxiolytic usage had significant effects on multiple ERG outcomes. Mixed model analysis was performed on data from dogs not receiving anxiolytic medications. RESULTS: In dogs not receiving anxiolytics, median age was 118 months (interquartile range 72-140 months, n = 77, 44 purebred, 33 mixed breed dogs). Age was significantly associated with prolonged peak times of a-waves (dark-adapted 3 and 10 cds/m2 flash p < 0.0001) and b-waves (cone flicker p = 0.03, dark-adapted 0.01 cds/m2 flash p = 0.001). Age was also significantly associated with reduced amplitudes of a-waves (dark-adapted 3 cds/m2 flash p < 0.0001, 10 cds/m2 flash p = 0.005) and b-waves (light-adapted 3 cds/m2 flash p < 0.0001, dark-adapted 0.01 cds/m2 flash p = 0.0004, 3 cds/m2 flash p < 0.0001, 10 cds/m2 flash p = 0.007) and flicker (light-adapted 30 Hz 3 cds/m2 p = 0.0004). Within the Golden Retriever breed, these trends were matched in a cross-sectional analysis of 6 individuals that received no anxiolytic medication. CONCLUSIONS: Aged companion dogs have slower and reduced amplitude responses in both rod- and cone-mediated ERG. Consideration of anxiolytic medication use should be made when conducting ERG studies in dogs.

Therapeutic targeting of tumor-associated myeloid cells synergizes with radiation therapy for glioblastoma.

Tumor-associated myeloid cells (TAMCs) are key drivers of immunosuppression in the tumor microenvironment, which profoundly impedes the clinical response to immune-dependent and conventional therapeutic modalities. As a hallmark of glioblastoma (GBM), TAMCs are massively recruited to reach up to 50% of the brain tumor mass. Therefore, they have recently been recognized as an appealing therapeutic target to blunt immunosuppression in GBM with the hope of maximizing the clinical outcome of antitumor therapies. Here we report a nano-immunotherapy approach capable of actively targeting TAMCs in vivo. As we found that programmed death-ligand 1 (PD-L1) is highly expressed on glioma-associated TAMCs, we rationally designed a lipid nanoparticle (LNP) formulation surface-functionalized with an anti-PD-L1 therapeutic antibody (αPD-L1). We demonstrated that this system (αPD-L1-LNP) enabled effective and specific delivery of therapeutic payload to TAMCs. Specifically, encapsulation of dinaciclib, a cyclin-dependent kinase inhibitor, into PD-L1-targeted LNPs led to a robust depletion of TAMCs and an attenuation of their immunosuppressive functions. Importantly, the delivery efficiency of PD-L1-targeted LNPs was robustly enhanced in the context of radiation therapy (RT) owing to the RT-induced up-regulation of PD-L1 on glioma-infiltrating TAMCs. Accordingly, RT combined with our nano-immunotherapy led to dramatically extended survival of mice in 2 syngeneic glioma models, GL261 and CT2A. The high targeting efficiency of αPD-L1-LNP to human TAMCs from GBM patients further validated the clinical relevance. Thus, this study establishes a therapeutic approach with immense potential to improve the clinical response in the treatment of GBM and warrants a rapid translation into clinical practice.

Computed tomography findings in a cohort of 169 dogs with elbow dysplasia - a retrospective study.

BACKGROUND: Canine elbow dysplasia (CED) is a complex developmental skeletal disorder associated with a number of pathological conditions within the cubital joint. Because CED is a heritable disease, it is important to identify and remove the affected animals from breeding. The first objective of this study was to describe the prevalence of medial coronoid process disease (MCPD) without (MCD) or with (FMCP) fragmented medial coronoid process, osteochondrosis (OC) and/or osteochondritis dissecans (OCD), ununited anconeal process (UAP), radio-ulnar incongruence (INC R-U) and humero-ulnar incongruence (INC H-U) in dogs with the use of CT imaging. The second aim was to determine the influence of demographics on the prevalence of investigated pathologies in dogs with clinical evidence of elbow dysplasia. RESULTS: In this retrospective study, CT data records of 169 dogs of different breeds presented to the small animal veterinary clinic from 2012 to 2018 were included. 69.23% of dogs diagnosed with CED were young (≤ 2 years old). The mean age of dogs presented with INC R-U was 1.68 ± 1.82 years, while in dogs without INC R-U the mean age was 2.64 ± 2.59 years. The mean age of dogs with INC H-U was 1.94 ± 2.06 years, while without INC H-U 3.29 ± 2.09 years. Labrador Retrievers, German Shepherd and Bernese Mountain dogs were most frequently presented with CED-associated lameness. In 122 dogs OA of varying severity was found. CONCLUSION: INC H-U, FMCP and MCD were among the most frequently found components of CED found in the present study. OCD and UAP were the least frequently diagnosed. Dogs presented with INC R-U and INC H-U were significantly younger than dogs without these CED components. Boxers, Dog de Bordeaux, American Staffordshire terriers and mixed-breed dogs were diagnosed later in life than the other breeds. OA of varying severity was found in 72.18% of dogs. Males accounted for more than 75% of the study population.

Quality of Life of Patients After Laparoscopic Pyeloplasty Due to Ureteropelvic Junction Obstruction: A Long-Term Observation.

This study aims to define the quality of life (QoL) of patients who had undergone laparoscopic pyeloplasty due to ureteropelvic junction obstruction. The QoL was investigated in 26 patients after pyeloplasty, on average, at a 7.5-year follow-up. The operation was performed in a single center between 2002 and 2009 and its effectiveness was confirmed by diuretic renography. The QoL was assessed using the World Health Organization Quality of Life (WHOQOL-BREF) questionnaire. Additionally, we used an own questionnaire, created for this study, specifically assessing the health-related quality of life after pyeloplasty. Overall, 96% of patients were satisfied with the surgical procedure and all would agree to have another pyeloplasty procedure if needed. In one case, dissatisfaction was caused by persisting postoperative pain. All patients but one, dissatisfied due to persisting pain, reported that the postoperative pain intensity was not a problem that would impact the QoL or professional activity. We conclude that laparoscopic pyeloplasty did not adversely affect the patients' QoL, which might stem from beneficial functional outcomes making the patients satisfied with treatment results.

GCN2 is essential for CD8+ T cell survival and function in murine models of malignant glioma.

Amino acid deprivation is a strategy that malignancies utilize to blunt anti-tumor T-cell immune responses. It has been proposed that amino acid insufficiency in T-cells is detected by GCN2 kinase, which through phosphorylation of EIF2α, shuts down global protein synthesis leading to T-cell arrest. The role of this amino acid stress sensor in the context of malignant brain tumors has not yet been studied, and may elucidate important insights into the mechanisms of T-cell survival in this harsh environment. Using animal models of glioblastoma and animals with deficiency in GCN2, we explored the importance of this pathway in T-cell function within brain tumors. Our results show that GCN2 deficiency limited CD8+ T-cell activation and expression of cytotoxic markers in two separate murine models of glioblastoma in vivo. Importantly, adoptive transfer of antigen-specific T-cells from GCN2 KO mice did not control tumor burden as well as wild-type CD8+ T-cells. Our in vitro and in vivo data demonstrated that reduction in amino acid availability caused GCN2 deficient CD8+ T-cells to become rapidly necrotic. Mechanistically, reduced CD8+ T-cell activation and necrosis was due to a disruption in TCR signaling, as we observed reductions in PKCθ and phoshpo-PKCθ on CD8+ T-cells from GCN2 KO mice in the absence of tryptophan. Validating these observations, treatment of wild-type CD8+ T-cells with a downstream inhibitor of GCN2 activation also triggered necrosis of CD8+ T-cells in the absence of tryptophan. In conclusion, our data demonstrate the vital importance of intact GCN2 signaling on CD8+ T-cell function and survival in glioblastoma.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression.

Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood-brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

Adoptive Transfer of IL13Rα2-Specific Chimeric Antigen Receptor T Cells Creates a Pro-inflammatory Environment in Glioblastoma.

In order to fully harness the potential of immunotherapy with chimeric antigen receptor (CAR)-modified T cells, pre-clinical studies must be conducted in immunocompetent animal models that closely mimic the immunosuppressive malignant glioma (MG) microenvironment. Thus, the goal of this project was to study the in vivo fate of T cells expressing CARs specific for the MG antigen IL13Rα2 (IL13Rα2-CARs) in immunocompetent MG models. Murine T cells expressing IL13Rα2-CARs with a CD28.ζ (IL13Rα2-CAR.CD28.ζ) or truncated signaling domain (IL13Rα2-CAR.Δ) were generated by retroviral transduction, and their effector function was evaluated both in vitro and in vivo. IL13Rα2-CAR.CD28.ζ T cells' specificity toward IL13Rα2 was confirmed through cytokine production and cytolytic activity. In vivo, a single intratumoral injection of IL13Rα2-CAR.CD28.ζ T cells significantly extended the survival of IL13Rα2-expressing GL261 and SMA560 glioma-bearing mice; long-term survivors were resistant to re-challenge with IL13Rα2-negative and IL13Rα2-positive tumors. IL13Rα2-CAR.CD28.ζ T cells proliferated, produced cytokines (IFNγ, TNF-α), and promoted a phenotypically pro-inflammatory glioma microenvironment by inducing a significant increase in the number of CD4+ and CD8+ T cells and CD8α+ dendritic cells and a decrease in Ly6G+ myeloid-derived suppressor cells (MDSCs). Our data underline the significance of CAR T cell studies in immunocompetent hosts and further validate IL13Rα2-CAR T cells as an efficacious therapeutic strategy for MG.

Winning the race with aging: age-related changes in gait speed and its association with cognitive performance in dogs.

Introduction: In humans, gait speed is a crucial component in geriatric evaluation since decreasing speed can be a harbinger of cognitive decline and dementia. Aging companion dogs can suffer from age-related mobility impairment, cognitive decline and dementia known as canine cognitive dysfunction syndrome. We hypothesized that there would be an association between gait speed and cognition in aging dogs. Methods: We measured gait speed on and off leash in 46 adult and 49 senior dogs. Cognitive performance in senior dogs was assessed by means of the Canine Dementia Scale and a battery of cognitive tests. Results: We demonstrated that dogs' food-motivated gait speed off leash is correlated with fractional lifespan and cognitive performance in dogs, particularly in the domains of attention and working memory. Discussion: Food-motivated gait speed off leash represents a relatively easy variable to measure in clinical settings. Moreover, it proves to be a more effective indicator of age-related deterioration and cognitive decline than gait speed on leash.

[The usefulness of the polymeric "double J" catheter in ureteropelvic junction stenting after laparoscopic pyeloplasty]. / Skutecznosc szynowania polaczeniamiedniczkowo-moczowodowego polimerowym cewnikiem "double J" u chorych poddanych laparoskopowej pyeloplastyce.

OBJECTIVES: The aim of the study was to evaluate both the effectiveness of ureteropelvic junction stenting with the use of a polymeric "double J" stent after laparoscopic pyeloplasty, as well as intra- and postoperative complications of the procedure. MATERIAL AND METHODS: From October 2001 to November 2010 laparoscopic pyeloplasty was performer in 150 patients with primary UPJO. In all cases an attempt has been made to insert the stent into the operated kidney before the operation. All but one operations were performed using a transperitoneal approach. Anderson-Hynes pyeloplasty was carried out in 85 cases, Y-V plasty in 61 cases and Fenger plasty in 2 patients. Open conversion was performer in two cases. RESULTS: In 13 cases the complications observed in the postoperastive period were connected with the polymeric stent. In 12 patients the obstruction of the stent lead to massive urinary leakage and in 3 cases from this group urinoma developed. Urinoma was also observed in a patient, who was left without stenting because of the difficulties in inserting the stent to the operated kidney. In 10 cases the stent was successfully replaced but in 3 cases the placement of a percutaneous nephrostomy tube was necessary. It was left in place for 3 weeks. CONCLUSIONS: Our data indicate, that the stent obstruction is the main cause of postoperative complications in patients after laparoscopic pyeloplasty. So far no better method of upper urinary tract drainage after laparoscopic pyeloplasty has been worked out. Experimenting with various types of stents has been of no avail.

[The use of polymeric Hem-o-lock clips in laparoscopic radical nephrectomy]. / Zastosowanie polimerowych klipsów Hem-o-lock w laparoskopowej nefrektomii radykalnej.

OBJECTIVES: The aim of the study was to evaluate the usefulness and cost-effectiveness of polymeric Hem-o-lock clips during laparoscopic nephrectomy. The intra- and postoperative complications of the operation were assessed too. MATERIAL AND METHODS: From April 2011 through November 2011, 19 laparoscopic radical nephrectomies were performed. A preferred method to secure the renal vein was the use of polymeric Hem-o-lock clips. The renal artery was clipped by titanium clips. In five patients an Endo-GIA stapler was used to secure the renal pedicle. All procedures were carried out using a transperitoneal access. The perioperative data were analyzed retrospectively. RESULTS: No intraoperative complications associated with the use of Hem-o-lock clips were observed. The mean procedure time was 202 min. The average blood loss during the operation was 480 ml. No bleeding in the postoperative period was observed. The mean abdominal drain output was 65 ml per day. The mean time to drain removal was 3 days. The average hospital stay was 5-6 days. CONCLUSIONS: Using the polymeric Hem-o-lock clips is a safe, relatively easy and cheep way to close the renal vein during laparoscopic radical nephrectomy.

Relationship between hearing, cognitive function, and quality of life in aging companion dogs.

BACKGROUND: Elderly people with presbycusis are at higher risk for dementia and depression than the general population. There is no information regarding consequences of presbycusis in dogs. OBJECTIVE: Evaluate the relationship between cognitive function, quality of life, and hearing loss in aging companion dogs. ANIMALS: Thirty-nine elderly companion dogs. METHODS: Prospective study. Hearing was evaluated using brainstem auditory evoked response (BAER) testing. Dogs were grouped by hearing ability. Owners completed the canine dementia scale (CADES) and canine owner-reported quality of life (CORQ) questionnaire. Cognitive testing was performed, and cognitive testing outcomes, CADES and CORQ scores and age were compared between hearing groups. RESULTS: Nineteen dogs could hear at 50 dB, 12 at 70 dB, and 8 at 90 dB with mean ages (months) of 141 ± 14, 160 ± 16, and 172 ± 15 for each group respectively (P = .0002). Vitality and companionship CORQ scores were significantly lower as hearing deteriorated (6.6-5.4, 50-90 dB group, P = .03 and 6.9-6.2, 50-90 dB group, P = .02, respectively). Cognitive classification by CADES was abnormal in all 90 dB group dogs and normal in 3/12 70 dB group and 11/19 50 dB group dogs (P = .0004). Performance on inhibitory control, detour and sustained gaze tasks decreased significantly with hearing loss (P = .001, P = .008, P = .002, respectively). In multivariate analysis, higher CADES score was associated with worse hearing (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Presbycusis negatively alters owner-pet interactions and is associated with poor executive performance and owner-assessed dementia severity.

Use of Cognitive Testing, Questionnaires, and Plasma Biomarkers to Quantify Cognitive Impairment in an Aging Pet Dog Population.

BACKGROUND: Aging dogs may suffer from canine cognitive dysfunction syndrome (CCDS), a condition in which cognitive decline is associated with amyloid pathology and cortical atrophy. Presumptive diagnosis is made through physical examination, exclusion of systemic/metabolic conditions, and completion of screening questionnaires by owners. OBJECTIVE: This study aimed to determine whether cognitive function could be quantified in aging pet dogs, and to correlate cognitive testing with validated questionnaires and plasma neurofilament light chain (pNfL) concentration. METHODS: Thirty-nine dogs from fifteen breeds were recruited (9.3 to 15.3 years). Owners completed the Canine Dementia Scale (CADES) and Canine Cognitive Dysfunction Rating scale (CCDR). Executive control and social cues were tested, and pNfL was measured with single molecule array assay. Comparisons were made between cognitive testing scores, CADES, CCDR scores, and pNfL. RESULTS: CADES scoring classified five dogs as severe CCDS, six as moderate, ten as mild, and eighteen as normal. CCDR identified seven dogs at risk of CCDS and thirty-two as normal. Cognitive testing was possible in the majority of dogs, although severely affected dogs were unable to learn tasks. CADES score correlated with sustained attention duration (r = -0.47, p = 0.002), inhibitory control (r = -0.51, p = 0.002), detour (r = -0.43, p = 0.001), and pNfL (r = 0.41, p = 0.025). Concentration of pNfL correlated with inhibitory control (r = -0.7, p≤0.001). The CCDR scale correlated with performance on inhibitory control (r = -0.46, p = 0.005). CONCLUSION: Our findings suggest that a multi-dimensional approach using a combination of questionnaires, specific cognitive tests, and pNfL concentration can be used to quantify cognitive decline in aging pet dogs.

Plasma Amyloid Beta Concentrations in Aged and Cognitively Impaired Pet Dogs.

Longevity-associated neurological disorders have been observed across human and canine aging populations. Alzheimer's disease (AD) and canine cognitive dysfunction syndrome (CDS) represent comparable diseases affecting both species as they age. Translational diagnostic and therapeutic research is needed for these incurable diseases. The amyloid ß (Aß) peptide family are AD-associated peptides with identical amino acid sequences between dogs and humans. Plasma Aß42 concentration increases with age and decreases with AD in humans, and cerebrospinal fluid (CSF) concentration decreases in AD and correlates inversely with the amyloid load within the brain. Similarly, CSF Aß42 concentrations decrease in dogs with CDS but there is limited and conflicting information on plasma Aß42 concentrations in aging dogs and dogs with CDS. We measured plasma concentrations of Aß42 and Aß40 with an ultrasensitive single-molecule array assay (SIMOA) in a population of healthy aging dogs of different life stages (n = 36) and dogs affected with CDS (n = 11). In addition, the ratio of Aß42/ß40 was calculated. The mean plasma concentrations of Aß42 and Aß40 increased significantly with age (r2 = 0.27, p = 0.001; and r2 = 0.42, p < 0.001, respectively) and with life stage: puppy/junior group (0.43-2 years): 1.23 ± 0.95 and 38.26 ± 49.43 pg/mL; adult/mature group (2.1-9 years): 10.99 ± 5.45 and 131.05 ± 80.17 pg/mL; geriatric/senior group (9.3-14.5 years): 18.65 ± 16.65 and 192.88 ± 146.38 pg/mL, respectively. Concentrations of Aß42 and Aß40 in dogs with CDS (11.0-15.6 years) were significantly lower than age-matched healthy dogs at 11.61 ± 6.39 and 150.23 ± 98.2 pg/mL (p = 0.0048 and p = 0.001), respectively. Our findings suggest the dynamics of canine plasma amyloid concentrations are analogous to that found in aging humans with and without AD.

Learning curve or experience-related outcome: what really matters in paediatric laparoscopic pyeloplasty.

INTRODUCTION: The process of improving one's skills over time is called a "learning curve". This term has attracted great attention during the last decades, especially in relation to laparoscopic techniques. AIM: To assess the outcome of paediatric laparoscopic pyeloplasty (LP). MATERIAL AND METHODS: Retrospective analysis of the consecutive LPs. The inclusion criteria: (1) children aged < 18 years, (2) transperitoneal approach, and (3) the same operating paediatric urologist (RC). Patients with a history of any procedure on the upper urinary tract were excluded. Any surgical reintervention during follow-up was defined as a failure. The outcomes of LPs performed before 2012 (G1) were compared to those conducted between 2012 and 2016 (G2). Fisher's exact test was used for statistical analysis. RESULTS: Ninety patients met the inclusion criteria, and a total of 95 LPs were performed. The mean operation time was 155 min, and the mean hospitalisation period was 2.4 days. In G1, 19 patients underwent Anderson-Hynes LP, 16 had Fenger non-dismembered LP and two underwent vascular hitch. In G2, 54, 2 and 2 patients underwent these procedures, respectively. The overall success rate was 91.5%. There were six failures in G1 and three in G2 (p = 0.147). Of the Anderson-Hynes LPs, 1/19 in G1 and 3/58 in G2 required reintervention (p = 1). For Fenger LPs, this was 4/16 and 0/2, respectively (p = 1). Only one patient required reoperation after vascular hitch. CONCLUSIONS: The surgeons' learning curve reflects their experience with regard to the entire therapeutic process, but not exclusively their manual skills.

Plasma Neurofilament Light Chain as a Translational Biomarker of Aging and Neurodegeneration in Dogs.

Age is a primary risk factor for multiple comorbidities including neurodegenerative diseases. Pet dogs and humans represent two populations that have experienced a significant increase in average life expectancy over the last century. A higher prevalence of age-related neurodegenerative diseases has been observed across both species, and human diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), have canine analogs, canine cognitive dysfunction (CCD), and degenerative myelopathy (DM) respectively. In humans, protein biomarkers have proved useful in the prediction and diagnosis of neurodegeneration. Molecular signatures of many proteins are highly conserved across species. In this study, we explored the potential of the neuronal cytoskeletal protein neurofilament light chain (NfL) as a biomarker of neuro-aging in dogs using an ultrasensitive single-molecule array assay to measure plasma concentrations. Healthy dogs of different ages and dogs affected with CCD and DM were evaluated. The mean plasma NfL concentrations in the different age groups of the healthy population were as follows: 4.55 ± 1.70 pg/mL in puppy/junior group (0.43-2 years), 13.51 ± 6.8 pg/mL in adult/mature group (2.1-9 years), and 47.1 ± 12.68 pg/mL in geriatric/senior group (9.3-14.5 years). Concentrations in dogs with DM (7.5-12.6 years) and CCD (11.0-15.6 years) were 84.17 ± 53.57 pg/mL and 100.73 ± 83.72 pg/mL, respectively. Plasma NfL increases in an age-dependent manner and is significantly elevated in dogs diagnosed with neurodegenerative disease. This work identified plasma NfL as a key clinical index of neuro-aging and neurodegeneration in pet dogs. Our findings mirror recent reports from human neurodegenerative diseases.

BET inhibition increases ßIII-tubulin expression and sensitizes metastatic breast cancer in the brain to vinorelbine.

Metastases from primary breast cancer result in poor survival. ßIII-tubulin (TUBB3) has been established as a therapeutic target for breast cancer metastases specifically to the brain. In this study, we conducted a systematic analysis to determine the regulation of TUBB3 expression in breast cancer metastases to the brain and strategically target these metastases using vinorelbine (VRB), a drug approved by the U.S. Food and Drug Administration (FDA). We found that human epidermal growth factor receptor 2 (HER2) signaling regulates TUBB3 expression in both trastuzumab-sensitive and trastuzumab-resistant neoplastic cells. We further discovered that bromodomain and extra-terminal domain (BET) inhibition increases TUBB3 expression, rendering neoplastic cells more susceptible to apoptosis by VRB. Orthotopic xenograft assays using two different breast cancer cell models revealed a reduction in tumor volume with BET inhibition and VRB treatment. In addition, in vivo studies using a model of multiple brain metastasis (BM) showed improved survival with the combination of radiation + BET inhibitor (iBET-762) + VRB (75% long-term survivors, P < 0.05). Using in silico analysis and BET inhibition, we found that the transcription factor myeloid zinc finger-1 (MZF-1) protein binds to the TUBB3 promoter. BET inhibition decreases MZF-1 expression and subsequently increases TUBB3 expression. Overexpression of MZF-1 decreases TUBB3 expression and reduces BM in vivo, whereas its knockdown increases TUBB3 expression in breast cancer cells. In summary, this study demonstrates a regulatory mechanism of TUBB3 and provides support for an application of BET inhibition to sensitize breast cancer metastases to VRB-mediated therapy.

Management of crossing vessels in children and adults: A multi-center experience with the transperitoneal laparoscopic approach.

BACKGROUND: Crossing vessels (CVs) are common in older children and adults with hydronephrosis but no gold standard exists on how to treat this condition. The final decision is made intraoperatively by the surgeon. OBJECTIVES: To assess the outcome of the laparoscopic dismembered pyeloplasty with translocation of the CVs in children and adults. MATERIAL AND METHODS: Prospectively collected data from 3 departments was reviewed. Inclusion criteria were: 1) a transperitoneal laparoscopic approach; 2) dismembered pyeloplasty; and 3) the same operating pediatric urologist (RC) or urologist (TS). In the case of CVs, pyeloplasty with vessel transposition (children) or with cephalad translocation (adults) was performed. Forty-eight children and 41 adults met these criteria. Patients were divided into 4 groups: children with (group 1A) and without (group 1B) CVs, and adults with (group 2A) and without (group 2B) CVs. Any surgical reintervention at the uretero-pelvic junction (UPJ) was deemed a failure. RESULTS: The overall reintervention rate was 3/48 (6.25%) in children and 2/41 (4.9%) in adults (p > 0.05), and involved the following: 4 endopyelotomies and 1 redo pyeloplasty. Crossing vessels were identified in 28/48 (58%) children and 12/41 (29%) adults. The mean operation time was 152 min in group 1A and 161 min in group 2A (p > 0.5). Reintervention was needed in 2/28 patients in group 1A and in 1/12 patients in group 2A (p > 0.05). There was no difference in the failure rate between group 1A and group 1B, nor between group 2A and group 2B (p > 0.05). CONCLUSIONS: Crossing vessels should be meticulously looked for during pyeloplasty in older children and adults. Dismembered laparoscopic pyeloplasty (LP) with dorsal transposition or cephalad translocation are comparable methods in terms of success rate for the treatment of UPJ obstruction in these patients.

FABP7 is a key metabolic regulator in HER2+ breast cancer brain metastasis.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer patients is associated with increased incidence of breast cancer brain metastases (BCBM), but the mechanisms underlying this phenomenon remain unclear. Here, to identify brain-predominant genes critical for the establishment of BCBM, we conducted an in silico screening analysis and identified that increased levels of fatty acid-binding protein 7 (FABP7) correlate with a lower survival and higher incidence of brain metastases in breast cancer patients. We validated these findings using HER2+ BCBM cells compared with parental breast cancer cells. Importantly, through knockdown and overexpression assays, we characterized the role of FABP7 in the BCBM process in vitro and in vivo. Our results uncover a key role of FABP7 in metabolic reprogramming of HER2 + breast cancer cells, supporting a glycolytic phenotype and storage of lipid droplets that enable their adaptation and survival in the brain microenvironment. In addition, FABP7 is shown to be required for upregulation of key metastatic genes and pathways, such as integrins-Src and VEGFA, and for the growth of HER2+ breast cancer cells in the brain microenvironment in vivo. Together, our results support FABP7 as a potential target for the treatment of HER2+ BCBM.

HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma.

The mechanisms by which regulatory T cells (Tregs) migrate to and function within the hypoxic tumor microenvironment are unclear. Our studies indicate that specific ablation of hypoxia-inducible factor 1α (HIF-1α) in Tregs results in enhanced CD8+ T cell suppression versus wild-type Tregs under hypoxia, due to increased pyruvate import into the mitochondria. Importantly, HIF-1α-deficient Tregs are minimally affected by the inhibition of lipid oxidation, a fuel that is critical for Treg metabolism in tumors. Under hypoxia, HIF-1α directs glucose away from mitochondria, leaving Tregs dependent on fatty acids for mitochondrial metabolism within the hypoxic tumor. Indeed, inhibition of lipid oxidation enhances the survival of mice with glioma. Interestingly, HIF-1α-deficient-Treg mice exhibit significantly enhanced animal survival in a murine model of glioma, due to their stymied migratory capacity, explaining their reduced abundance in tumor-bearing mice. Thus HIF-1α acts as a metabolic switch for Tregs between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression.

Local Application of Autologous Platelet-Rich Fibrin Patch (PRF-P) Suppresses Regulatory T Cell Recruitment in a Murine Glioma Model.

The immunosuppressive microenvironment is one of the major factors promoting the growth of glioblastoma multiforme (GBM). Infiltration of CD4+CD25+Foxp3+ regulatory T cells (Tregs) into the tumor microenvironment plays a significant role in the suppression of the anti-tumor immunity and portends a dismal prognosis for patients. Glioma-mediated secretion of chemo-attractant C-C motif ligand 2 and 22 (CCL2/22) has previously been shown by our group to promote Treg migration in vitro. In this study, we show that a local implantation of platelet-rich fibrin patch (PRF-P) into the brain of GL261 glioma-bearing mice prolonged the survival of affected animals by 42.85% (p = 0.0011). Analysis performed on brain tumor tissue harvested from PRF-P-treated mice revealed a specific decrease in intra-tumoral lymphocytes with a preferential depletion of immunosuppressive Tregs. Importantly, co-culture of GL261 or chemo-attractants (CCL2/22) with PRF-P abrogated Treg migration. Pharmacological blockade of the CCL2/22 interaction with their receptors potentiated the inhibitory effect of PRF-P on Tregs recruitment in culture. Moreover, our findings revealed the soluble CD40 ligand (sCD40L) as a major Treg inhibitory player produced by activated platelets entrapped within the fibrin matrix of the PRF-P. Blockade of sCD40L restored the migratory capacity of Tregs, emphasizing the role of PRF-P in preventing the Treg migration to glioma tissue. Our findings highlight autologous PRF-P as a personalized, Treg-selective suppression platform that can potentially supplement and enhance the efficacy of glioma therapies.