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Purpose: Immunotherapy promises unprecedented benefits to patients with cancer. However, the majority of cancer types, including high-risk neuroblastoma, remain immunologically unresponsive. High-intensity focused ultrasound (HIFU) is a noninvasive technique that can mechanically fractionate tumors, transforming immunologically “cold” tumors into responsive “hot” tumors. Experimental Design: We treated Results: Combining HIFU with αCTLA-4 and αPD-L1 significantly enhances antitumor response, improving survival from 0% to 62.5%. HIFU alone causes upregulation of splenic and lymph node NK cells and circulating IL2, IFNγ, and DAMPs, whereas immune regulators like CD4+Foxp3+, IL10, and VEGF-A are significantly reduced. HIFU combined with checkpoint inhibitors induced significant increases in intratumoral CD4+, CD8α+, and CD8α+CD11c+ cells, CD11c+ in regional lymph nodes, and decrease in circulating IL10 compared with untreated group. We also report significant abscopal effect following unilateral treatment of mice with large, established bilateral tumors using HIFU and checkpoint inhibitors compared with tumors treated with HIFU or checkpoint inhibitors alone (61.1% survival, P < 0.0001). This combination treatment significantly also induces CD4+CD44+hiCD62L+low and CD8α+CD44+hiCD62L+low population and is adoptively transferable, imparting immunity, slowing subsequent de novo tumor engraftment. Conclusions: Mechanical fractionation of tumors using HIFU can effectively induce immune sensitization in a previously unresponsive murine neuroblastoma model and promises a novel yet efficacious immunoadjuvant modality to overcome therapeutic resistance.

Summary Acute hypoxia (HX) causes extensive cellular damage in the developing human cerebral cortex. We found increased expression of activated-EGFR in affected cortical areas of neonates with HX and investigated its functional role in the piglet, which displays a highly evolved, gyrencephalic brain, with a human-like maturation pattern. In the piglet, HX-induced activation of EGFR and Ca2+/calmodulin kinase IV (CaMKIV) caused cell death and pathological alterations in neurons and glia. EGFR blockade inhibited CaMKIV activation, attenuated neuronal loss, increased oligodendrocyte proliferation, and reversed HX-induced astrogliosis. We performed for the first time high-throughput transcriptomic analysis of the piglet cortex to define molecular responses to HX and to uncover genes specifically involved in EGFR signaling in piglet and human brain injury. Our results indicate that specific molecular responses modulated by EGFR may be targeted as a therapeutic strategy for HX injury in the neonatal brain., Graphical Abstract, Highlights • EGFR mediates the effects of neonatal hypoxia in piglet and human cerebral cortex • EGFR inhibition reverses pathological changes induced by hypoxia in piglet cortex • EGFR blockage prevents hypoxia-induced transcriptomic changes in piglet cortex • EGFR-associated pathways are therapeutic targets in neonatal hypoxic injury, Porcine Molecular Biology; Developmental Neuroscience; Transcriptomics

RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap: 5 kb), large (Neb: 22 kb), and very large (Ttn: 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type-specific differential expression of these novel transcripts. The improved transcript identification and quantification shown by our approach removes previous impediments to studies aimed at quantitative differential expression of ultralong transcripts.

RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon-junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap−5kb), large (nebulin - 22 kb) and very-large (titin - 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type specific differential expression of these novel transcripts. The improved transcript identification and quantification demonstrated by our approach removes previous impediments to studies aimed at quantitative differential expression of ultra-long transcripts.

BackgroundPreterm infants are frequently exposed to intermittent hypoxia (IH) associated with apnea and periodic breathing that may result in inflammation and brain injury that later manifests as cognitive and executive function deficits. We used a rodent model to determine whether early postnatal exposure to IH would result in inflammation and brain injury.MethodsRat pups were exposed to IH from P2 to P12. Control animals were exposed to room air. Cytokines were analyzed in plasma and brain tissue at P13 and P18. At P20-P22, diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) were performed.ResultsPups exposed to IH had increased plasma Gro/CXCL1 and cerebellar IFN-γ and IL-1β at P13, and brainstem enolase at P18. DTI showed a decrease in FA and AD in the corpus callosum (CC) and cingulate gyrus, and an increase in RD in the CC. MRS revealed decreases in NAA/Cho, Cr, Tau/Cr, and Gly/Cr; increases in TCho and GPC in the brainstem; and decreases in NAA/Cho in the hippocampus.ConclusionsWe conclude that early postnatal exposure to IH, similar in magnitude to that experienced in human preterm infants, is associated with evidence for proinflammatory changes, decreases in white matter integrity, and metabolic changes consistent with hypoxia.

Cytokines have been proposed as mediators of neonatal brain injury via neuroinflammatory pathways triggered by hypoxia-ischemia. Limited data are available on cytokine profiles in larger cohorts of newborns with hypoxic-ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH). Serum cytokines interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-α, and interferon-γ were measured in newborns with HIE at 24 and 72 h of TH. Differences between infants with favorable (survivors with mild/no magnetic resonance imaging (MRI) injury) vs. adverse outcome (death or moderate/severe MRI injury) were compared using mixed models to adjust for covariates. Data from 36 term newborns with HIE (favorable outcome: n = 20, adverse outcome: n = 16) were evaluated. Cytokines IL-1β, IL-2, IL-6, IL-8, IL-10, and IL-13 were elevated in the adverse relative to favorable outcome group at 24 h. IL-6 remained significantly elevated in the adverse outcome group at 72 h. IL-6 and IL-10 remained significantly associated with outcome group after controlling for covariates. Inflammatory cytokines are elevated in HIE newborns with brain injury by MRI. In particular, IL-6 and IL-10 were associated with adverse outcomes after controlling for baseline characteristics and severity of presentation. These data suggest that cytokine response may identify infants in need of additional neuroprotective interventions.

Background Real-time quantitative PCR (RTqPCR) detects minimal residual disease (MRD) in few leukemia patients after transplant because of the rarity of available targets. We sought to quantify the commonly expressed tumor-associated antigens (TAA) commonly expressed by high risk leukemias: WT1, PRAME, and BIRC5 (survivin), by meausring circulating mRNA in peripheral blood. We employed droplet digital PCR (ddPCR) as a novel, minimally invasive method that enables the quantitation of target nucleic acids with high precision, accuracy, and low specimen input. Objective Demonstrate that simultaneous quantification of WT1, PRAME, and BIRC5 mRNA levels in peripheral blood by ddPCR correlates with leukemia burden in HSCT recipients. Design/Methods RNA was isolated from PBMCs and bone marrow from HSCT patients on an IRB-approved study, and from healthy controls. RainDance ddPCR was first optimized to simultaneously quantitate levels of the TAAs in tumor cell lines (hepatoblastoma and K-562 myelogenous leukemia) and then used to assay mRNA levels in marrow and peripheral blood of patients with acute leukemia and healthy controls. Expression of WT1, PRAME, BIRC5, and ABL1 (homeostatic gene) were assessed simultaneously in all samples with RNA integrity number (RIN) ≥6. Results/discussion Following assay optimization the median levels in healthy controls were determined for each TAA/ABL1 ratio (n=20). Results were consistent with previously published values for qPCR. Marrow levels of TAA mRNA exceeded peripheral blood (mean fold increase 8.4; range: 1.6-17), consistent with TAA/ABL1 ratio changes reflecting marrow leukemia burden. Disease response directly correlated with blood ddPCR TAA/ABL1 levels (ID#1 Day 21: BIRC5: 0.97 (relapse) and Day 100: BIRC5: 0.096 (remission); ID#2 day 60: BCR-ABL-neg/ BIRC5: 0.19 (CNS relapse) and Day 100: BCR-ABL 0.4%/ BIRC5: 0.77 (progression). Of those patients with active leukemia, 93% (13/14) were positive for at least one TAA. In summary, we have developed a new method for monitoring MRD in leukemia (patent submitted) and present preliminary data that ddPCR measurement of TAA/ABL1 ratio may correlate with circulating leukemia burden in HSCT recipients.

Background MicroRNAs (miRNAs) are noncoding RNA molecules that play important roles in the pathogenesis of various kidney diseases. We investigated whether patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) have distinct circulating and urinary miRNA expression profiles that could lead to potential development of noninvasive biomarkers of the disease. Materials and methods Exosome miRNAs were extracted from plasma and urine samples of patients with primary FSGS (n = 16) or MCD (n = 5) and healthy controls (n = 5). Differences in miRNA abundance were examined using Affymetrix GeneChip miRNA 3.0 arrays. QRT-PCR was used to validate the findings from the array. Results Comparison analysis of FSGS versus MCD revealed 126 and 155 differentially expressed miRNAs in plasma and in urine, respectively. Only 38 of these miRNAs were previously cited, whereas the remaining miRNAs have not been described. Comparison analysis showed that a significant number of miRNAs were downregulated in both plasma and urine samples of patients with FSGS compared to those with MCD. Plasma levels of miR-30b, miR-30c, miR-34b, miR-34c and miR-342 and urine levels of mir-1225-5p were upregulated in patients with MCD compared to patients with FSGS and controls (P

MicroRNAs (miRNAs) are noncoding RNAs that regulate posttranscriptional gene expression. In this study we characterized the circulating and urinary miRNA pattern associated with reduced glomerular filtration rate, using Affymetrix GeneChip miR 4.0 in 28 patients with chronic kidney disease (CKD). Top miRNA discoveries from the human studies were validated in an Alb/TGFβ mouse model of CKD, and in rat renal proximal tubular cells (NRK52E) exposed to TGFβ1. Plasma and urinary levels of procollagen III N-terminal propeptide and collagen IV were elevated in patients with decreased estimated glomerular filtration rate (eGFR). Expression of 384 urinary and 266 circulatory miRNAs were significantly different between CKD patients with eGFR ≥30 vs. −1·1.73 m−2. Pathway analysis mapped multiple miRNAs to TGFβ signaling-related mRNA targets. Specifically, Let-7a was significantly downregulated, and miR-130a was significantly upregulated, in urine of patients with eGFR

Multiple sclerosis is a chronic disease of the central nervous system characterized by an autoimmune inflammatory reaction that leads to axonal demyelination and tissue damage. Glucocorticoids, such as prednisolone, are effective in the treatment of multiple sclerosis in large part due to their ability to inhibit pro-inflammatory pathways (e.g., NFκB). However, despite their effectiveness, long-term treatment is limited by adverse side effects. VBP15 is a recently described compound synthesized based on the lazeroid steroidal backbone that shows activity in acute and chronic inflammatory conditions, yet displays a much-reduced side effect profile compared to traditional glucocorticoids. The purpose of this study was to determine the effectiveness of VBP15 in inhibiting inflammation and disease progression in experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of multiple sclerosis. Our data show that VBP15 is effective at reducing both disease onset and severity. In parallel studies, we observed that VBP15 was able to inhibit the production of NFκB-regulated pro-inflammatory transcripts in human macrophages. Furthermore, treatment with prednisolone-but not VBP15-increased expression of genes associated with bone loss and muscle atrophy, suggesting lack of side effects of VBP15. These findings suggest that VBP15 may represent a potentially safer alternative to traditional glucocorticoids in the treatment of multiple sclerosis and other inflammatory diseases.

Dominant mutations in the valosin-containing protein (VCP) gene cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, which is characterized by progressive muscle weakness, dysfunction in bone remodeling, and frontotemporal dementia. More recently, VCP has been linked to 2% of familial amyotrophic lateral sclerosis cases. VCP plays a significant role in a plethora of cellular functions including membrane fusion, transcription activation, nuclear envelope reconstruction, postmitotic organelle reassembly, and cell cycle control. To elucidate the pathological mechanisms underlying the VCP disease progression, we have previously generated a VCP(R155H/+) mouse model with the R155H mutation. Histological analyses of mutant muscle showed vacuolization of myofibrils, centrally located nuclei, and disorganized muscle fibers. Global expression profiling of VCP(R155H/+) mice using gene annotations by DAVID identified key dysregulated signaling pathways including genes involved in the physiological system development and function, diseases and disorders, and molecular and cellular functions. There were a total of 212 significantly dysregulated genes, several of which are involved in the regulation of proteasomal function and NF-κB signaling cascade. Findings of the gene expression study were validated by using quantitative reverse transcriptase polymerase chain reaction analyses to test genes involved in various signaling cascades. This investigation reveals the importance of the VCP(R155H/+) mouse model in the understanding of cellular and molecular mechanisms causing VCP-associated neurodegenerative diseases and in the discovery of novel therapeutic advancements and strategies for patients suffering with these debilitating disorders.

Prader-Willi syndrome (PWS) is a genetic disorder caused by deficiency of imprinted gene expression from the paternal chromosome 15q11-15q13 and clinically characterized by neonatal hypotonia, short stature, cognitive impairment, hypogonadism, hyperphagia, morbid obesity, and diabetes. Previous clinical studies suggest that a defect in energy metabolism may be involved in the pathogenesis of PWS. We focused our attention on the genes associated with energy metabolism and found that there were 95 and 66 mitochondrial genes differentially expressed in PWS muscle and brain, respectively. Assessment of enzyme activities of mitochondrial oxidative phosphorylation complexes in the brain, heart, liver, and muscle were assessed. We found the enzyme activities of the cardiac mitochondrial complexes II+‫III were up-regulated in the PWS imprinting center deletion mice compared to the wild-type littermates. These studies suggest that differential gene expression, especially of the mitochondrial genes may contribute to the pathophysiology of PWS.

Summary Because molecular mechanisms underlying refractory focal epilepsy are poorly defined, we performed transcriptome analysis on human epileptogenic tissue. Compared with controls, expression of Circadian Locomotor Output Cycles Kaput (CLOCK) is decreased in epileptogenic tissue. To define the function of CLOCK, we generated and tested the Emx-Cre; Clockflox/flox and PV-Cre; Clockflox/flox mouse lines with targeted deletions of the Clock gene in excitatory and parvalbumin (PV)-expressing inhibitory neurons, respectively. The Emx-Cre; Clockflox/flox mouse line alone has decreased seizure thresholds, but no laminar or dendritic defects in the cortex. However, excitatory neurons from the Emx-Cre; Clockflox/flox mouse have spontaneous epileptiform discharges. Both neurons from Emx-Cre; Clockflox/flox mouse and human epileptogenic tissue exhibit decreased spontaneous inhibitory postsynaptic currents. Finally, video-EEG of Emx-Cre; Clockflox/flox mice reveals epileptiform discharges during sleep and also seizures arising from sleep. Altogether, these data show that disruption of CLOCK alters cortical circuits and may lead to generation of focal epilepsy.

Inflammation has long been implicated in secondary tissue damage after spinal cord injury (SCI). Our previous studies of inflammatory gene expression in rats after SCI revealed two temporally correlated clusters: the first was expressed early after injury and the second was up-regulated later, with peak expression at 1-2 weeks and persistent up-regulation through 6 months. To further address the role of inflammation after SCI, we examined inflammatory genes in a second species, mice, through 28 days after SCI. Using anchor gene clustering analysis, we found similar expression patterns for both the acute and chronic gene clusters previously identified after rat SCI. The acute group returned to normal expression levels by 7 days post injury. The chronic group, which included C1qB, p22(phox) and galectin-3, showed peak expression at 7 days and remained up-regulated through 28 days. Immunohistochemistry and western blot analysis showed that the protein expression of these genes was consistent with the mRNA expression. Further exploration of the role of one of these genes, galectin-3, suggests that galectin-3 may contribute to secondary injury. In summary, our findings extend our prior gene profiling data by demonstrating the chronic expression of a cluster of microglial associated inflammatory genes after SCI in mice. Moreover, by demonstrating that inhibition of one such factor improves recovery, the findings suggest that such chronic up-regulation of inflammatory processes may contribute to secondary tissue damage after SCI, and that there may be a broader therapeutic window for neuroprotection than generally accepted.

Galectins are pleiotropic carbohydrate-binding lectins involved in inflammation, growth/differentiation, and tissue remodeling. The functional role of galectins in amyotrophic lateral sclerosis (ALS) is unknown. Expression studies revealed increases in galectin-1 mRNA and protein in spinal cords from SOD1(G93A) mice, and in galectin-3 and -9 mRNAs and proteins in spinal cords of both SOD1(G93A) mice and sporadic ALS patients. As the increase in galectin-3 appeared in early presymptomatic stages and increased progressively through to end stage of disease in the mouse, it was selected for additional study, where it was found to be mainly expressed by microglia. Galectin-3 antagonists are not selective and do not readily cross the blood-brain barrier; therefore, we generated SOD1(G93A)/Gal-3(-/-) transgenic mice to evaluate galectin-3 deletion in a widely used mouse model of ALS. Disease progression, neurological symptoms, survival, and inflammation were assessed to determine the effect of galectin-3 deletion on the SOD1(G93A) disease phenotype. Galectin-3 deletion did not change disease onset, but resulted in more rapid progression through functionally defined disease stages, more severely impaired neurological symptoms at all stages of disease, and expiration, on average, 25 days earlier than SOD1(G93A)/Gal-3(+/+) cohorts. In addition, microglial staining, as well as TNF-α, and oxidative injury were increased in SOD1(G93A)/Gal-3(-/-) mice compared with SOD1(G93A)/Gal-3(+/+) cohorts. These data support an important functional role for microglial galectin-3 in neuroinflammation during chronic neurodegenerative disease. We suggest that elevations in galectin-3 by microglia as disease progresses may represent a protective, anti-inflammatory innate immune response to chronic motor neuron degeneration.

The development of a tissue-engineered vascular graft (TEVG) holds great promise for advancing the field of cardiac surgery. Despite the successful translation of this technology, previous reports identify the primary mode of graft failure as stenosis secondary to intimal hyperplasia. MicroRNAs (miRNAs) regulate gene expression by interfering with mRNA function and recent research has suggested miRNA as a potential therapeutic target. The role of miRNAs in TEVGs during neotissue formation is currently unknown. In this study, we investigated if miRNAs regulate the inhibition of graft stenosis. Biodegradable PGA-P(LA/CL) scaffolds were implanted as inferior vena cava interposition grafts in a murine model (n = 14). Mice were sacrificed 14 days following implantation and TEVGs were harvested for histological analysis and miRNA profiling using Affymetrix miRNA arrays. Graft diameters were measured histologically, and the largest grafts (patent group) and smallest grafts (stenosed group) were profiled (n = 4 for each group). Cell population in each graft was analyzed with immunohistochemistry using antismooth muscle actin (SMA) and antimacrophage (F4/80) antibodies. The graft diameter was significantly greater in the patent group (0.63 ± 0.06 mm) than in the stenosed group (0.17 ± 0.06 mm) (p < 0.01). Cell proliferation was significantly greater in the stenosed grafts than in patent grafts (p < 0.01: SMA [187 ± 11 vs. 77 ± 8 cells] vs. p = 0.025: F4/80 [245 ± 23 vs. 187 ± 11 cells]). MiRNA array of 1416 genes showed that in stenosed grafts, mir-451, mir-338, and mir-466 were downregulated and mir-154 was upregulated. Mir-451 exhibited the greatest difference in expression between stenosed and patent grafts by -3.1-fold. Significant negative correlation was found between the expression of mir-451 and cell proliferation (SMA: r = -0.86, p = 0.003; F4/80: r = -0.89, p = 0.001). Our data, along with previous evidence that mir-451 regulates tumor suppressor genes, suggest that downregulation of mir-451 promotes acute proliferation of macrophages and smooth muscle cells, thereby inducing TEVG stenosis. Adequate expression of mir-451 may be critical for improving TEVG patency.

The dual role of microglia in cytotoxicity and neuroprotection is believed to depend on the specific, temporal expression of microglial-related genes. To better clarify this issue, we used high-density oligonucleotide microarrays to examine microglial gene expression after spinal cord injury (SCI) in rats. We compared expression changes at the lesion site, as well as in rostral and caudal regions after mild, moderate, or severe SCI. Using microglial-associated anchor genes, we identified two clusters with different temporal profiles. The first, induced by 4 h postinjury to peak between 4 and 24 h, included interleukin-1β, interleukin-6, osteopontin, and calgranulin, among others. The second was induced 24 h after SCI, and peaked between 72 h and 7 days; it included C1qB, Galectin-3, and p22phox. These two clusters showed similar expression profiles regardless of injury severity, albeit with slight decreases in expression in mild or severe injury vs. moderate injury. Expression was also decreased rostral and caudal to the lesion site. We validated the expression of selected cluster members at the mRNA and protein levels. In addition, we demonstrated that stimulation of purified microglia in culture induces expression of C1qB, Galectin-3, and p22phox. Finally, inhibition of p22phox activity within microglial cultures significantly suppressed proliferation in response to stimulation, confirming that this gene is involved in microglial activation. Because microglial-related factors have been implicated both in secondary injury and recovery, identification of temporally distinct clusters of genes related to microglial activation may suggest distinct roles for these groups of factors. © 2005 Wiley-Liss, Inc.

Activity-based therapy (ABT) focuses on regaining motor and sensory function below the level of the lesion in persons with a spinal cord injury (SCI). This is accomplished through repetitive training of specific motor tasks. Research has shown that ABT may increase neuroplasticity in the rat and human spinal cord.The primary aim of this study was to examine acute alterations in neuroplasticity-related proteins during ABT in persons with SCI.Volunteers were current participants in an ABT program and consisted of 12 men and 3 women (age, 31.8 ± 10.9 years) with chronic SCI (injury duration, 63.9 ± 54.4 months). A single 2-hour bout of ABT consisted of standing load bearing, body weight-supported treadmill training, whole body vibration, and functional electrical stimulation. Blood samples were obtained at baseline and immediately after completion of each modality to determine serum levels of brain-derived neurotrophic factor (BDNF), prolactin, and cortisol.One-way analysis of variance (ANOVA) with repeated measures was used to examine differences in proteins over time. Results revealed baseline levels of BDNF (2.37 ± 1.41 ng/mL) that were lower than previous research has demonstrated in persons with SCI. No change in BDNF or cortisol was found, although prolactin was significantly reduced in response to ABT.Despite the length of the bout, acute changes in BDNF were not observed. Whether different intensities or modalities of ABT may promote acute increases in serum BDNF in individuals with SCI remains to be determined and further study is merited.

Background Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. Methods A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. Results A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in subjects where host defenses were initiated. Conclusions Counter to clinical belief, healthy urine is not sterile. The healthy urine microbiome is characterized by a preponderance of Lactobacillales in women and Corynebacterium in men. The presence and duration of NB and method of urinary catheterization alter the healthy urine microbiome. An integrated approach of 16S rDNA sequencing with metaproteomics improves our understanding of healthy urine and facilitates a more personalized approach to prevention and treatment of infection.

Inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder caused by mutations in the Valosin‐containing protein (VCP) gene on chromosome 9p12–13. Patients demonstrate limb girdle muscle weakness, which eventually progresses to involve respiratory muscles, and death from respiratory and cardiac failure. This is the first investigation to analyze key molecular mediators and signaling cascades in skeletal muscle causing myopathy by global gene microarray in hopes of understanding the dysregulated genes and molecular mechanisms underlying IBMPFD and the hope of finding novel therapeutic targets. We determined expression profiles using Human Genome Array microarray technology in Vastus lateralis muscles from patients and their first‐degree relatives. We analyzed gene annotations by Database for Annotation, Visualization and Integration Discovery and identified differentially dysregulated genes with roles in several novel biological pathways, including regulation of actin cytoskeleton, ErbB signaling, cancer, in addition to regulation of autophagy, and lysosomal signaling, known disrupted pathways in VCP disease. In this report, we present data from the first global microarray analyzing IBMPFD patient muscles and elucidating dysregulated pathways to further understand the pathogenesis of the disease and discover potential therapeutics. Clin Trans Sci 2012; Volume #: 1–9

Background Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI. Methods Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression. Results Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma. Conclusions These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.

The dysferlin deficient SJL/J mouse strain is commonly used to study dysferlin deficient myopathies. Therefore, we systematically evaluated behavior in relatively young (9–25 weeks) SJL/J mice and compared them to C57BL6 mice to determine which functional end points may be the most effective to use for preclinical studies in the SJL/J strain. SJL/J mice had reduced body weight, lower open field scores, higher creatine kinase levels, and less muscle force than did C57BL6 mice. Power calculations for expected effect sizes indicated that grip strength normalized to body weight and open field activity were the most sensitive indicators of functional status in SJL/J mice. Weight and open field scores of SJL/J mice deteriorated over the course of the study, indicating that progressive myopathy was ongoing even in relatively young (

Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.

Duchenne muscular dystrophy (DMD; dystrophin-deficiency) causes dilated cardiomyopathy in the second decade of life in affected males. We studied the dystrophin-deficient mouse heart ( mdx ) using high-frequency echocardiography, histomorphometry, and gene expression profiling. Heart dysfunction was prominent at 9–10months of age and showed significantly increased LV internal diameter (end systole) and decreased posterior wall thickness. This cardiomyopathy was associated with a 30% decrease in shortening fraction. Histologically, there was a 10-fold increase in connective tissue volume (fibrosis). mRNA profiling with RT-PCR validation showed activation of key pro-fibrotic genes, including Nox4 and Lox. The Nox gene family expression differed in mdx heart and skeletal muscle, where Nox2 was specifically induced in skeletal muscle while Nox4 was specifically induced in heart. This is the first report of an altered profibrotic gene expression profile in cardiac tissue of dystrophic mice showing echocardiographic evidence of cardiomyopathy.

Changes in gene expression contribute to pathophysiological alterations following spinal cord injury (SCI). We examined gene expression over time (4 h, 24 h, 7 days) at the impact site, as well as rostral and caudal regions, following mild, moderate, or severe contusion SCI in rats. High-density oligonucleotide microarrays were used that included ∼27,000 genes/ESTs (Affymetrix RG-U34; A, B and C arrays), together with multiple analyses (MAS 5.0, dChip). Alterations after mild injury were relatively rapid (4 and 24 h), whereas they were delayed and prolonged after severe injury (24 h and 7 days). The number and magnitude of gene expression changes were greatest at the injury site after moderate injury and increased in rostral and caudal regions as a function of injury severity. Sham surgery resulted in expression changes that were similar to mild injury, suggesting the importance of using time-linked surgical controls as well as naive animals for these kinds of studies. Expression of many genes and ESTs was altered; these were classified functionally based on ontology. Overall representation of these functional classes varied with distance from the site of injury and injury severity, as did the individual genes that contributed to each functional class. Different clustering approaches were used to identify changes in neuronal-specific genes and several transcription factors that have not previously been associated with SCI. This study represents the most comprehensive evaluation of gene expression changes after SCI to date. The results underscore the power of microarray approaches to reveal global genomic responses as well as changes in particular gene clusters and/or families that may be important in the secondary injury cascade.

Spinal cord injury (SCI) results in delayed biochemical changes that contribute to secondary tissue damage and dysfunction. The microglial-induced inflammatory response, long implicated in secondary tissue damage after SCI, includes release of various factors known to be toxic to neurons. However, the response of microglia and their subsequent role remains controversial and poorly understood. The present study evaluated the acute and chronic gene expression profiles in rats after traumatic SCI. Using anchor genes selected from the literature because of their relationship to microglial-induced inflammation, high density microarray expression profiles were examined after contusion. Two temporally correlated clusters were identified: one was expressed immediately after injury, peaking between 4 and 24 hours and declining to baseline or below baseline levels; the second was expressed chronically after injury, and remained elevated up to 6 months after injury. Expression profiles for genes of each group were confirmed using real-time quantitative PCR, immunoblotting, and immunohistochemistry. One of the genes chronically expressed from 24 hours to 6 months postinjury was p22phox, a component of the NADPH oxidase enzyme. To investigate the role of this chronically expressed gene after SCI and the effect of modification of its activity, the NADPH oxidase inhibitor apocynin was added to purified microglia cultures and microglial activation after LPS stimulation was investigated. Inhibition of p22phox/NADPH oxidase resulted in significant suppression of microglial proliferation, nitric oxide production, and microglial-induced neurotoxicity. This study demonstrates the potential utility of microarray technology in identifying possible targets for future therapeutics. Additionally, it identifies p22phox and NADPH as potential components of microglial-induced neurotoxicity, with implications for neuroprotective therapy.

Summary The hypothalamus integrates information required for the production of a variety of innate behaviors such as feeding, mating, aggression, and predator avoidance. Despite an extensive knowledge of hypothalamic function, how embryonic genetic programs specify circuits that regulate these behaviors remains unknown. Here, we find that in the hypothalamus the developmentally regulated homeodomain-containing transcription factor Dbx1 is required for the generation of specific subclasses of neurons within the lateral hypothalamic area/zona incerta (LH) and the arcuate (Arc) nucleus. Consistent with this specific developmental role, Dbx1 hypothalamic-specific conditional-knockout mice display attenuated responses to predator odor and feeding stressors but do not display deficits in other innate behaviors such as mating or conspecific aggression. Thus, activity of a single developmentally regulated gene, Dbx1 , is a shared requirement for the specification of hypothalamic nuclei governing a subset of innate behaviors. Video Abstract