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Although the early diagnosis of gastric cancer provides the opportunity for curative endoscopic resection, comprehensive screening endoscopy would be invasive and expensive. To date, there is a complete absence of clinically useful gastric cancer biomarkers. With the goal of discovering noninvasive biomarkers for the early diagnosis of gastric cancer, we have conducted a case–control study using urine samples from individuals with gastric cancer versus healthy control samples. Of the enrolled 106 patients from September, 2012 to April, 2013, a cohort of 70 patients composed of 35 patients with gastric cancer and 35 age- and sex-matched healthy controls was analyzed. The gastric cancer group was composed of stage IA of 62.9% (22/35). The urinary levels of MMP-9/NGAL complex (uMMP-9/NGAL) and ADAM12 (uADAM12) were significantly higher in the gastric cancer group compared with the healthy control group as determined by monospecific ELISAs (uMMP-9/NGAL: median, 85 pg/mL vs. 0 pg/mL; P = 0.020; uADAM12: median, 3.35 ng/mL vs. 1.44 ng/mL; P < 0.001). Multivariate analysis demonstrated that both uMMP-9/NGAL and uADAM12 were significant, independent diagnostic biomarkers for gastric cancer. Moreover, MMP-9/NGAL activity was significantly elevated as determined by gelatin zymography. The combination of uMMP-9/NGAL with uADAM12 distinguished between control samples and gastric cancer samples with an AUC of 0.825 (P < 0.001) in an ROC analysis. Significantly, immunohistochemical analyses demonstrated a high coexpression of MMP-9 and NGAL (P < 0.001) and high expression of ADAM12 (P < 0.001) in gastric cancer tissues compared with adjacent normal tissues (N = 35). In summary, uMMP-9/NGAL and uADAM12 are potential noninvasive biomarkers for gastric cancer, including early-stage disease. Cancer Prev Res; 8(3); 240–8. ©2015 AACR.

Supplementary Figure S2. Representative immunohistochemical images of MMP-9 and NGAL in early gastric cancer without urinary MMP-9/NGAL complex.

Supplementary Figure S1. Angiogenesis array analyses of urine sample.

De novo mutations causing dysfunction of the ATP1A3 gene, which encodes the α3 subunit of Na+/K+-ATPase pump expressed in neurons, result in alternating hemiplegia of childhood (AHC). AHC manifests as paroxysmal episodes of hemiplegia, dystonia, behavioral abnormalities, and seizures. The first aim of this study was to characterize a novel knock-in mouse model (Atp1a3E815K+/-, Matoub, Matb+/-) containing the E815K mutation of the Atp1a3 gene recognized as causing the most severe and second most common phenotype of AHC with increased morbidity and mortality as compared to other mutations. The second aim was to investigate the effects of flunarizine, currently the most effective drug used in AHC, to further validate our model and to help address a question with significant clinical implications that has not been addressed in prior studies. Specifically, many E815K patients have clinical decompensation and catastrophic regression after discontinuing flunarizine therapy; however, it is not known whether this is congruent with the natural course of the disease and is a result of withdrawal from an acute beneficial effect, withdrawal from a long-term protective effect or from a detrimental effect of prior flunarizine exposure. Our behavioral and neurophysiological testing demonstrated that Matb+/- mice express a phenotype that bears a strong resemblance to the E815K phenotype in AHC. In addition, these mice developed spontaneous seizures with high incidence of mortality and required fewer electrical stimulations to reach the kindled state as compared to wild-type littermates. Matb+/- mice treated acutely with flunarizine had reduction in hemiplegic attacks as compared with vehicle-treated mice. After withdrawal of flunarizine, Matb+/- mice that had received flunarizine did neither better nor worse, on behavioral tests, than those who had received vehicle. We conclude that: 1) Our mouse model containing the E815K mutation manifests clinical and neurophysiological features of the most severe form of AHC, 2) Flunarizine demonstrated acute anti-hemiplegic effects but not long-term beneficial or detrimental behavioral effects after it was stopped, and 3) The Matb+/- mouse model can be used to investigate the underlying pathophysiology of ATP1A3 dysfunction and the efficacy of potential treatments for AHC.

To determine the neuropsychological abnormalities that occur in alternating hemiplegia of childhood (AHC) and report on our experience in managing them.Patients underwent evaluations according to our standardized AHC pathway. Data were entered into our prospective AHC database and then analyzed.Of the cohort of 25 consecutive patients (ages 15mo-42y), eight had initial chief complaints about cognition, 14 language, five attention, and 11 behavior. As compared to population norms means, neuropsychological and behavioral assessment tools (including Child Behavior Checklist, Vineland Adaptive Behavior Scales, Peabody Picture Vocabulary, and Wechsler Intelligence Quotient tests) showed significant impairments in multiple domains: cognition, expressive and receptive language, executive function/attention, and behavior (p0.05 in all comparisons). Evaluations generated management recommendations in all patients. Twenty had neuropsychiatric diagnoses: 10 attention-deficit/hyperactivity disorder (ADHD), seven disruptive behavior, and three anxiety disorder. Eight out of nine patients with ADHD who were prescribed medications responded to pharmacotherapy.Patients with AHC have developmental difficulties related to impairments in multiple neuropsychological domains. This supports the hypothesis that the underlying AHC pathophysiology involves diffuse neuronal dysfunction. Testing generated recommendations to help manage these difficulties. Patients with AHC also have a range of neuropsychiatric diagnoses, the most common being ADHD which responds to pharmacotherapy.Patients with alternating hemiplegia of childhood (AHC) have developmental difficulties with underlying neuropsychological impairments. The findings in this study are consistent with an underlying AHC pathophysiology which involves diffuse neuronal, probably largely GABAergic, dysfunction. Patients with AHC have a range of neuropsychiatric diagnoses, the most common being attention-deficit/hyperactivity disorder.PERFILES COGNITIVOS, ADAPTATIVOS Y DE COMPORTAMIENTO Y MANEJO DE LA HEMIPLEJIA ALTERNA EN LA INFANCIA: OBJETIVO: Determinar las anormalidades neuropsicológicas que ocurren en la hemiplejia alterna en la infancia (AHC) y reportar nuestra experiencia en su manejo.Los pacientes fueron evaluados de acuerdo a nuestra evaluación estandarizado de AHC. Los datos fueron introducidos en nuestra base de datos de AHC prospectiva y luego analizados.De la cohorte de 25 pacientes consecutivos (edades 15 meses - 42 años), ocho tenían desafíos iniciales sobre cognición, 14 de lenguaje, cinco de atención y 11 de comportamiento. Comparadas con la población tipica, las herramientas de evaluación neuropsicológica y de comportamiento (incluyendo Child Behavior Checklist, Vineland Adaptive Behavior Scales, Peabody Picture Vocabulary, y Wechsler Intelligence Quotient Tests) mostraron discapacidades significativas en múltiples áreas: cognición, lenguaje expresivo y receptivo, atención y función ejecutiva, y comportamiento (p0.05 en todas las comparaciones). Las evaluaciones generaron recomendaciones de manejo para todos los pacientes. Veinte tenían diagnósticos neuropsiquiátricos: 10 desorden de hiperactividad y déficit de atención (TDAH), siete comportamientos disruptivos, y tres desorden de ansiedad. Ocho de cada nueve pacientes con TDAH a los cuales se les prescribió medicación respondieron a la terapia farmacológica.Los pacientes con AHC tienen dificultades en el desarrollo relacionadas a discapacidades en múltiples áreas neuropsicológicas. Esto apoyo la hipótesis que la fisiopatología AHC subyacente involucra disfunción neuronal difusa. Las evaluaciones generaron recomendaciones para ayudar a manejar esas dificultades. Los pacientes con AHC también tienen un rango de diagnósticos neuropsiquiátricos, siendo TDAH la más común que responde a terapia farmacológica.PERFIL COGNITIVO, ADAPTATIVO E COMPORTAMENTAL DA HEMIPLEGIA ALTERNANTE DA INFÂNCIA: OBJETIVO: Determinar anormalidades neuropsicológicas que ocorrem na hemiplegia alternante da infância (HAI) e relatar nossa experiência com o manejo dos mesmos. MÉTODO: Pacientes passaram por avaliações de acordo com nossa rotina para HAI. Os dados foram entrados em uma base de dados prospectiva e analisados. RESULTADOS: Da coorte de 25 pacientes consecutivos (idades 15m-42a), oito tiveram queixas iniciais importantes sobre cognição, 14 de linguagem, 5 de atenção e 8 de comportamento. Em comparação com valores normativos da população, os instrumentos de avaliação neuropsicológica e comportamental (incluindo Checklist de Comportamento da criança, Escalas Vineland de Comportamento Adaptativo, testes Peabody de Vocabulário pictórico e Quociente de inteligiencia Wechsler) mostraram alterações significativas em múltiplos domínios: cognição, linguagem expressiva e receptiva, função executiva/atenção, e comportamento (p0,05 em todas as comparações). As avaliações geraram recomendações de manejo em todos os pacientes. Vinte tiveram diagnósticos neuropsiquiátricos: 10 com transtorno de déficit de atenção e hiperatividade (TDAH), sete comportamento disruptivo, e três transtorno de ansiedade. Oito dos nove pacientes com TDAH que foram prescritos medicação responderam a farmacoterapia. INTERPRETAÇÃO: Pacientes com HAI têm dificuldades neurodesenvolvimentais relacionadas a deficiências em múltiplos domínios neuropsicológicos. Isso suporta a hipótese de que a fisiopatologia básica da HAI envolve disfunção neuronal difusa. As avaliações geraram recomendações que ajudaram a manejar estas dificuldades. Pacientes com HAI também têm uma variedade de diagnósticos neuropsiquiátricos, o mais comum sendo TDAH que responde a farmacoterapia.

Objective Na+ /K+ -ATPase dysfunction, primary (mutation) or secondary (energy crisis, neurodegenerative disease) increases neuronal excitability in the brain. To evaluate the mechanisms underlying such increased excitability we studied mice carrying the D801N mutation, the most common mutation causing human disease, specifically alternating hemiplegia of childhood (AHC) including epilepsy. Because the gene is expressed in all neurons, particularly γ-aminobutyric acid (GABA)ergic interneurons, we hypothesized that the pathophysiology would involve both pyramidal cells and interneurons and that fast-spiking interneurons, which have increased firing rates, would be most vulnerable. Methods We performed extracellular recordings, as well as whole-cell patch clamp recordings from pyramidal cells and interneurons, in the CA1 region on hippocampal slices. We also performed immunohistochemistry from hippocampal sections to count CA1 pyramidal cells as well as parvalbumin-positive interneurons. In addition, we performed video-electroencephalography (EEG) recordings from the dorsal hippocampal CA1 region. Results We observed that juvenile knock-in mice carrying the above mutation reproduce the human phenotype of AHC. We then demonstrated in the CA1 region of these mice the following findings as compared to wild type: (1) Increased number of spikes evoked by electrical stimulation of Schaffer collaterals; (2) equalization by bicuculline of the number of spikes induced by Schaffer collateral stimulation; (3) reduced miniature, spontaneous, and evoked inhibitory postsynaptic currents, but no change in excitatory postsynaptic currents; (4) robust action potential frequency adaptation in response to depolarizing current injection in CA1 fast-spiking interneurons; and (5) no change in the number of pyramidal cells, but reduced number of parvalbumin positive interneurons. Significance Our data indicate that, in our genetic model of Atp1α3 mutation, there is increased excitability and marked dysfunction in GABAergic inhibition. This supports the performance of further investigations to determine if selective expression of the mutation in GABAergic and or glutamatergic neurons is necessary and sufficient to result in the behavioral phenotype.

Hemimegalencephaly is known to occur in Proteus syndrome, but has not been reported, to our knowledge, in the other PTEN mutation-related syndrome of Bannayan-Riley-Ruvalcaba. Here, we report a patient with Bannayan-Riley-Ruvalcaba syndrome who also had hemimegalencephaly and in whom the hemimegalencephaly was evident well before presentation of the characteristic manifestations of Bannayan-Riley-Ruvalcaba syndrome. An 11-year-old boy developed drug-resistant focal seizures on the fifth day of life. MRI revealed left hemimegalencephaly. He later showed macrocephaly, developmental delay, athetotic quadriplegic cerebral palsy, and neuromuscular scoliosis. Freckling of the penis, which is characteristic of Bannayan-Riley-Ruvalcaba syndrome, was not present at birth but was observed at 9 years of age. Gene analysis revealed a c.510 T>G PTEN mutation. This patient and his other affected family members, his father and two siblings, were started on the tumour screening procedures recommended for patients with PTEN mutations. This case highlights the importance of early screening for PTEN mutations in cases of hemimegalencephaly not otherwise explained by another disorder, even in the absence of signs of Proteus syndrome or the full manifestations of Bannayan-Riley Ruvalcaba syndrome.

Purpose To report novel clinical manifestations of KCNA2 mutation related epileptic encephalopathy. Methods Blood samples were sent for whole exome and Sanger sequencing. Seizure types were characterized by clinical criteria and EEG recording. Results KCNA2 mutations have been reported in 10 cases who presented with focal, absence, generalized tonic-clonic or myoclonic astatic seizures. Here we describe 3 patients with previously unreported, more severe manifestations. Patient 1 is a 5 year-old male with a c.1214 C > T (p.Pro405Leu) mutation, previously reported to be disease causing. He presented at 1year of age with focal seizures and subsequently developed electrical status epilepticus of sleep at age 3. The latter finding to our knowledge has never been reported in patients with KCNA2 mutations. Patient 2 is a 7 year-old female with a novel c.1195 G > A (p.Val399Met) mutation not previously described. She presented with intermittent then continuous polymyoclonus and myoclonic-astatic and generalized tonic clonic seizures. Continuous polymyoclonus is another new manifestation in patients with KCNA2 mutations. Patient 3 is a 23 year-old male with a c.889C > T (p.Arg297Trp) mutation not previously described. He presented at 4 years of age with generalized tonic clonic seizures and later developed recurrent refractory status epilepticus episodes at ages 19, 22 and 23 years, the latter being a novel manifestation in patients with KCNA2 mutations. Conclusion We identified 3 patients with KCNA2 mutations with novel characteristics, including electrical status epilepticus of sleep, continuous polymyoclonus and status epilepticus. These results expand KCNA2 mutation epileptic manifestations to include more severe, previously unreported phenotypes.

ObjectiveTo report our experience and investigate 5 original hypotheses: (1) multiple types of epileptic seizures occur in alternating hemiplegia of childhood (AHC), and these can be the initial presentation; (2) epileptiform abnormalities often appear well after clinical seizures; (3) nonepileptic reduced awareness spells (RAS) occur frequently; (4) epilepsy is commonly drug resistant but may respond to vagal nerve stimulation (VNS); and (5) status epilepticus (SE) is common and is usually refractory and recurrent.MethodsWe analyzed a cohort of 51 consecutive patients with AHC.ResultsThirty-two of 51 patients had epilepsy: 18 focal seizures, frontal more frequently than temporal, and then posterior. Eleven had primary generalized seizures (tonic-clonic, myoclonic, and/or absence). Epileptic seizures preceded other AHC paroxysmal events in 8 (lag 5.63 ± 6.55 months; p = 0.0365). In 7 of 32, initial EEGs were normal, with the first epileptiform EEG lagging behind by 3.53 ± 4.65 years (p = 0.0484). RAS occurred equally in patients with epilepsy (16 of 32) and patients without epilepsy (10 of 19, p = 1.0). Twenty-eight patients had video-EEG; captured RAS showed no concomitant EEG changes. Nineteen patients (59%) were drug resistant. VNS resulted in >50% reduction in seizures in 5 of 6 (p < 0.04). Twelve patients (38%) had SE (9 of 12 multiple episodes), refractory/superrefractory in all (p < 0.001), and 4 of 12 had regression after SE.ConclusionsEpilepsy in AHC can be focal or generalized. Epileptic seizures may be the first paroxysmal symptom. EEG may become epileptiform only on follow-up. Epilepsy, although frequently drug resistant, can respond to VNS. RAS are frequent and nonepileptic. SE often recurs and is usually refractory/superrefractory. Our observations are consistent with current data on AHC-ATP1A3 pathophysiology.

NaWe performed extracellular recordings, as well as whole-cell patch clamp recordings from pyramidal cells and interneurons, in the CA1 region on hippocampal slices. We also performed immunohistochemistry from hippocampal sections to count CA1 pyramidal cells as well as parvalbumin-positive interneurons. In addition, we performed video-electroencephalography (EEG) recordings from the dorsal hippocampal CA1 region.We observed that juvenile knock-in mice carrying the above mutation reproduce the human phenotype of AHC. We then demonstrated in the CA1 region of these mice the following findings as compared to wild type: (1) Increased number of spikes evoked by electrical stimulation of Schaffer collaterals; (2) equalization by bicuculline of the number of spikes induced by Schaffer collateral stimulation; (3) reduced miniature, spontaneous, and evoked inhibitory postsynaptic currents, but no change in excitatory postsynaptic currents; (4) robust action potential frequency adaptation in response to depolarizing current injection in CA1 fast-spiking interneurons; and (5) no change in the number of pyramidal cells, but reduced number of parvalbumin positive interneurons.Our data indicate that, in our genetic model of Atp1α3 mutation, there is increased excitability and marked dysfunction in GABAergic inhibition. This supports the performance of further investigations to determine if selective expression of the mutation in GABAergic and or glutamatergic neurons is necessary and sufficient to result in the behavioral phenotype.

Although the early diagnosis of gastric cancer provides the opportunity for curative endoscopic resection, comprehensive screening endoscopy would be invasive and expensive. To date, there is a complete absence of clinically useful gastric cancer biomarkers. With the goal of discovering noninvasive biomarkers for the early diagnosis of gastric cancer, we have conducted a case–control study using urine samples from individuals with gastric cancer versus healthy control samples. Of the enrolled 106 patients from September, 2012 to April, 2013, a cohort of 70 patients composed of 35 patients with gastric cancer and 35 age- and sex-matched healthy controls was analyzed. The gastric cancer group was composed of stage IA of 62.9% (22/35). The urinary levels of MMP-9/NGAL complex (uMMP-9/NGAL) and ADAM12 (uADAM12) were significantly higher in the gastric cancer group compared with the healthy control group as determined by monospecific ELISAs (uMMP-9/NGAL: median, 85 pg/mL vs. 0 pg/mL; P = 0.020; uADAM12: median, 3.35 ng/mL vs. 1.44 ng/mL; P < 0.001). Multivariate analysis demonstrated that both uMMP-9/NGAL and uADAM12 were significant, independent diagnostic biomarkers for gastric cancer. Moreover, MMP-9/NGAL activity was significantly elevated as determined by gelatin zymography. The combination of uMMP-9/NGAL with uADAM12 distinguished between control samples and gastric cancer samples with an AUC of 0.825 (P < 0.001) in an ROC analysis. Significantly, immunohistochemical analyses demonstrated a high coexpression of MMP-9 and NGAL (P < 0.001) and high expression of ADAM12 (P < 0.001) in gastric cancer tissues compared with adjacent normal tissues (N = 35). In summary, uMMP-9/NGAL and uADAM12 are potential noninvasive biomarkers for gastric cancer, including early-stage disease. Cancer Prev Res; 8(3); 240–8. ©2015 AACR.

Purpose Gain-of-function mutations in the KCNT1 gene have been reported in a number of drug resistant epilepsy syndromes including Epilepsy of Infancy with Migrating Focal Seizures. Quinidine, a potassium channel blocker, has been proposed as a potential therapeutic agent with only a few patients reported in the literature to have received it. Here we report 3 additional children, with such KCNT1 mutations and refractory seizures, who received quinidine therapy. Methods Retrospective chart review of 3 children with KCNT1 mutations, of ages 3 months, 9 years and 13 years old. Video-EEG documented seizure type and frequency. Seizure frequency was compared before and after quinidine initiation. We then analyzed seizure response (defined as > 50% reduction in seizure frequency) as it related to age in our 3 reported children, an additional 2 previously seen by us in our center, and an additional 3 reported in the literature (total 8 cases). Results In our report, the 3-month-old infant responded to quinidine, while the two older children did not. Using a cutoff of 4 years of age, review of the total of 8 cases, five from our center, revealed that all patients younger than 4 years responded to quinidine (4/4), while none of the ones older than 4 years did (0/4). Conclusion The above-mentioned findings support performance of prospective controlled studies of quinidine efficacy in children with KCNT1 gain-of-function mutations that control for age as a possible variable affecting response.

Notwithstanding remarkable progress in vascular network engineering, implanted bioengineered microvessels largely fail to form anastomoses with the host vasculature. Here, we demonstrate that implants containing assembled human vascular networks (A-Grafts) fail to engraft due to their inability to engage non-inflammatory host neutrophils upon implantation into mice. In contrast, unassembled vascular cells (U-Grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-Grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-Grafts reengaged neutrophils and enhanced revascularization in A-Grafts, a process that was recapitulated by blocking Notch signaling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues.

The idiopathic nature of Urological Chronic Pelvic Pain Syndrome (UCPPS), which encompasses interstitial cystitis/bladder pain syndrome (IC/BPS) and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), has prompted an intense search for clinical biomarkers. Identification of clinically relevant, validated biologic markers of UCPPS has great potential to inform our understanding of UCPPS pathophysiology, development, and progression, and ultimately clinical management. To date, the cause of this syndrome is unknown and there is no ability to determine who is at risk of progression or who will respond to specific therapy. Accurate noninvasive tests, based on objective, specific, and definable levels of validated biomarkers, are essential to improve and standardized clinical guidelines and for more targeted management strategies based on patient profiles. The MAPP Research Network was established by the NIDDK of the NIH and represents a comprehensive approach to the study of UCPPS 1, 2. A major goal of this initiative is to better understand pathophysiology and identify potential therapeutic targets. In this MAPP Network study, we have utilized a biologically-driven, candidate biomarker discovery strategy, grounded in the basic biochemistry and physiology of this syndrome, to evaluate candidate non-invasive biomarkers of UCPPS. Reviews of the literature have revealed a number of candidate protein biomarker targets. Increased VEGF levels have been detected in bladder biopsy samples from patients with IC compared to controls, with levels correlating with pain severity 3, 4. Prior studies also suggest an important connection between neovascularization and IC, in that new blood vessels in biopsy samples of IC patients showed significantly lower levels of pericyte coverage of the nascent endothelium, evidence consistent with the known association between high levels of VEGF and immature vessel formation 3. Moreover, VEGF originally discovered as VPF (Vascular Permeability Factor), 5, 6 is the most potent endogenous vascular permeability factor and may mediate the dysregulated vascular permeability postulated to be important in UCPPS 7–9. Given these associations, we chose to include VEGF and its receptor VEGFR1, as biomarker candidates. Inflammation has been suggested to be an underlying pathophysiologic mechanism in UCPPS 10 and increased concentrations of pro-inflammatory cytokines (IL-1β, TNF-α, IL-8) have been reported in patients with UCPPS as compared with controls 11, 12. In this respect, MMPs are of particular interest. Originally thought to be associated exclusively with tissue remodeling and destruction, it is now widely appreciated that MMPs play a role in a variety of biologic processes including cytokine and growth factor release, tumor growth and progression, angiogenesis and a number of inflammatory conditions 13–17. During the inflammatory response, MMPs and their complexes are released from connective tissue cells in response to pro-inflammatory cytokines. Upregulation of MMP activity results in the recruitment of pro-inflammatory cells to the site of tissue injury. MMPs have also been implicated in the regulation of the immune response because of their ability to cleave inflammatory mediators and stimulate the clearance of inflammatory cells 18–21. Given that MMPs may play an important role in UCPPS as a function of the inflammatory phenotype, abnormal vasculature and the dysregulation of ECM (extracellular matrix) turnover present in this and other potentially related diseases we included these proteins in our study. NGAL, also known as Lipocalin-2 (Lcn2), is upregulated in numerous chronic and acute inflammatory conditions, including chronic kidney disease, ulcerative colitis and myocardial infarction 22, 23. There exists an intricate interplay between NGAL and other inflammatory cytokines as well, such that NGAL upregulation can be triggered by multiple inflammatory cytokines, including TNF-α, IL-1β and IL-17 22, 23. Moreover, NGAL itself is a modulator of the levels of other inflammatory cytokines, as well as of the behavior of inflammatory leukocytes 24–26. In our laboratory, we have demonstrated that NGAL is a significant stimulator of VEGF levels in breast tumor cells and can also independently induce the epithelial to mesenchymal transition in breast cancer, as well as being a noninvasive biomarker of this disease 27–29. NGAL can also form a complex with MMP-9, representing a distinct protein complex referred to as MMP-9/NGAL complex. We have previously shown that when NGAL complexes with MMP-9, it protects MMP-9 from degradation preserving its enzyme activity 16, 30. The MMP-9/NGAL complex has also been detected in urine samples from patients with various diseases. Based on these findings, we chose to measure NGAL as well as MMP-9/NGAL complex in the urine of individuals with UCPPS and correlate them with clinical symptoms. Here, we report sex-specific comparisons of candidate biomarkers among UCPPS and control cohorts to gain new insights into underlying pathophysiology. In addition, associations of biomarkers with symptom severity within the UCPPS cohort are examined. We anticipate that the identified biological markers may serve as potential candidates for further clinical evaluation, including in the development of new treatment strategies directed toward novel targets, as objective measures for patient classification schemes, and evaluation of clinical outcomes, as well as further characterization of underlying mechanism.

We examined symptom variability in men and women with urological chronic pelvic pain syndrome. We describe symptom fluctuations as related to early symptom regression and its effect on estimated 1-year symptom change. We also describe a method to quantify patient specific symptom variability.Symptoms were assessed biweekly in 424 subjects with urological chronic pelvic pain syndrome during 1 year. To evaluate the impact of early symptom regression subjects were classified as improved, no change or worse according to the rate of change using 1) all data, 2) excluding week 0 and 3) excluding weeks 0 and 2. Patient specific, time varying variability was calculated at each interval using a sliding window approach. Patients were classified as high, medium or low variability at each time and ultimately as high or low variability overall based on the variability for the majority of contacts.Prior to excluding early weeks to adjust for early symptom regression 25% to 38% and 5% to 6% of patients were classified as improved and worse, respectively. After adjustment the percent of patients who were improved or worse ranged from 15% to 25% and 6% to 9%, respectively. High and low variability phenotypes were each identified in 25% to 30% of participants.Patients with urological chronic pelvic pain syndrome show symptom variability. At study enrollment patients had worse symptoms on average, resulting in a regression effect that influenced the estimated proportion of those who were improved or worse. Prospective studies should include a run-in period to account for regression to the mean and other causes of early symptom regression. Further, symptom variability may be quantified and used to characterize longitudinal symptom profiles of urological chronic pelvic pain syndrome.

INTRODUCTION AND OBJECTIVES: Stress urinary incontinence, a major type of urinary incontinence, increases with age and is often developed after partum injury. Low intensity pulsed ultrasound (LIPUS) has been investigated in the treatment of many diseases showing its ability of restoring soft tissue injury. We investigated the therapeutic effect of low intensity pulsed ultrasound in stress urinary incontinence. METHODS: Thirty-two Sprague Dawley rats in SUI group underwent vaginal distension (VD) and bilateral ovariectomy mimicking partum injury. Eight rats served as mock operation control. Eight rats each in SUI group was treated with low-dosage LESW (0.03 mJ/mm2), medium-dosage LESW (0.06 mJ/mm2), or high-dosage LESW (0.09 mJ/mm2). The rest eight rats served as none-treatment group. For functional study, leak point pressure test (LPP) was performed 2 weeks after the last LESW. Masson trichrome staining was performed to validate the pathological changes. RESULTS: The LPP was restored in medium-dosage LESW and high-dosage LESW groups, but not in low-dosage LESW group. More robust striated muscle regeneration was found in these two group comparing with the none-treatment group. CONCLUSIONS: LIPUS ameliorate the symptom of SUI via activating striated muscle regeneration.

Helicobacter pylori infection is associated with severe chronic inflammation, yet the host immune response is rarely able to clear the bacterium. Thymus derived lymphocyte populations such as T helper 1, T helper 17, and T regulatory cells are known to play important roles in the chronicity of H. pylori infection as well as contributing to ongoing gastric pathology. It is yet to be established how these immune cell populations interact in the gastric environment during H. pylori infection. Mouse models of infection offer an opportunity to investigate these interactions in detail. Flow cytometric analysis provides excellent lymphocyte characterization due to its high specificity, sensitivity and potential to perform multiple simultaneous measurements. However, this requires a viable enriched single cell suspension after adequate tissue dissociation, which poses a challenge due to the heterogeneity of gastric tissue. We have evaluated several isolation techniques and have optimized a protocol to isolate and enrich lymphocytes from the H. pylori-infected murine stomach. EDTA/DTT followed by Collagenase IV digestion successfully dissociates an average of 1 × 10⁷ cells per mouse. Further enrichment using Lympholyte M gradient yields on average 4 × 106 CD45+ lymphocytes per stomach. Following isolation we compared lymphocyte stimulation by CD3/CD28, phorbol 12-myristate 13-acetate (PMA) and ionomycin or H. pylori lysate and determined that CD3/CD28 effectively induces stimulation of IFNγ and IL 17A, but impairs Foxp3 expression. Using an optimized protocol we observed a 2-fold increase of CD8+ IFNγ-expressing lymphocytes localized specifically to the gastric compartment during H. pylori infection. The mechanisms of H. pylori immunopathogenesis are still considered enigmatic, therefore this optimized protocol can help delineate further novel immune cell targets that mediate H. pylori-induced pathology and identify the correlates of immunity for vaccine development.

Background The objective of this study was to discover and to validate novel noninvasive biomarkers that distinguish between benign prostate hyperplasia (BPH) and localized prostate cancer (PCa), thereby helping to solve the diagnostic dilemma confronting clinicians who treat these patients. Methods Quantitative iTRAQ LC/LC/MS/MS analysis was used to identify proteins that are differentially expressed in the urine of men with BPH compared with those who have localized PCa. These proteins were validated in 173 urine samples from patients diagnosed with BPH (N = 83) and PCa (N = 90). Multivariate logistic regression analysis was used to identify the predictive biomarkers. Results Three proteins, β2M, PGA3, and MUC3 were identified by iTRAQ and validated by immunoblot analyses. Univariate analysis demonstrated significant elevations in urinary β2M (P

Ovarian cancer cells, both primary and metastatic, reside in an environment rich in adipocytes. Adipocytes have been shown to promote ovarian cancer metastasis by secreting chemoattractant cytokines and to stimulate growth by directly transferring fatty acids into cancer cells. In addition to metastasis, another key reason for the high mortality rate among ovarian cancer patients is chemoresistance. Whether adipocytes have any effects on ovarian cancer chemoresistance remains unknown. To address this question, we isolated conditioned media from human adipocytes and found that the conditioned media increased ovarian cancer cell survival in the presence of chemotherapeutic drugs. We confirmed the protective effects of adipocytes using different human ovarian cancer cell lines (CAOV3, OVCAR5, OVCAR8 and SKOV3) and multiple chemotherapy drugs (cisplatin, paclitaxel and doxorubicin). Conditioned media dialyzed using a 10kDa cut-off membrane displayed the same protection against chemotherapy drugs, suggesting that proteins, not lipids, are more likely to mediate the pro-survival effects of adipocytes. Adiponectin and Leptin are two of the most upregulated proteins during adipocyte differentiation. However, inhibition of these two proteins, either through siRNA silencing or antibody neutralization, did not diminish the pro-survival effects of adipocytes. We also investigated the downstream signaling pathways and found that Akt was activated by adipocyte-conditioned media and was critical for adipocyte-induced effects given that treating ovarian cancer cells with Akt pathway inhibitor LY294002 abolished the pro-survival effects of the adipocytes. Taken together, these data demonstrate that secreted factors from adipocytes enhance the survival of ovarian cancer cells treated with chemotherapeutic drugs by activating the Akt pathway and suggest that adipocytes and associated factors may be important mediators of ovarian cancer chemoresistance. (This work is supported by the Weitzman Family Fund.) Citation Format: Jiang Yang, Monisha Sachdev, Marsha Moses. Adipocytes promote ovarian cancer cell survival in the presence of chemotherapeutic drugs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A11.