Initial sensitivity to psychostimulants can predict subsequent use and abuse in humans. Acute locomotor activation in response to psychostimulants is commonly used as an animal model of initial drug sensitivity and has been shown to have a substantial genetic component. Identifying the specific genetic differences that lead to phenotypic differences in initial drug sensitivity can advance our understanding of the processes that lead to addiction. Phenotyping inbred mouse strain panels are frequently used as a first step for studying the genetic architecture of complex traits. We assessed locomotor activation following a single, acute 20 mg/kg dose of cocaine (COC) in males from 45 inbred mouse strains and observed significant phenotypic variation across strains indicating a substantial genetic component. We also measured levels of COC, the active metabolite, norcocaine and the major inactive metabolite, benzoylecgonine, in plasma and brain in the same set of inbred strains. Pharmacokinetic (PK) and behavioral data were significantly correlated, but at a level that indicates that PK alone does not account for the behavioral differences observed across strains. Phenotypic data from this reference population of inbred strains can be utilized in studies aimed at examining the role of psychostimulant-induced locomotor activation on drug reward and reinforcement and to test theories about addiction processes. Moreover, these data serve as a starting point for identifying genes that alter sensitivity to the locomotor stimulatory effects of COC.
Cocaine; inbred strains; locomotor; mice; pharmacokinetics
Succinate dehydrogenase (SDH) deficient renal carcinoma has been accepted as a provisional entity in the 2013 International Society of Urological Pathology Vancouver Classification. To further define its morphologic and clinical features, we studied a multi-institutional cohort of 36 SDH deficient renal carcinomas from 27 patients, including 21 previously unreported cases.
We estimate that 0.05-0.2% of all renal carcinomas are SDH deficient. Mean patient age at presentation was 37 years (range, 14 to 76 yrs), with a slight male predominance (M:F=1.7:1). 26% of patients had bilateral tumors. 34 (94%) of tumors demonstrated the previously reported morphology at least focally, which included: solid or focally cystic growth, uniform cytology with eosinophilic flocculent cytoplasm, intracytoplasmic vacuolations and inclusions, and round to oval low-grade nuclei. All 17 patients who underwent genetic testing for mutation in the SDH subunits demonstrated germline mutations (16 in SDHB and 1 in SDHC). 9 of 27 (33%) patients developed metastatic disease, 2 of them after prolonged follow-up (5.5 and 30 years). 7 of 10 patients (70%) with high-grade nuclei metastasized as did all 4 patients with coagulative necrosis. 2 of 17 (12%) patients with low-grade nuclei metastasized and both had unbiopsied contralateral tumors which may have been the origin of the metastatic disease.
In conclusion, SDH-deficient renal carcinoma is a rare and unique type of renal carcinoma, exhibiting stereotypical morphologic features in the great majority of cases and showing a strong relationship with SDH germline mutation. Although this tumor may undergo de-differentiation and metastasize, sometimes after a prolonged delay, metastatic disease is rare in the absence of high grade nuclear atypia or coagulative necrosis.
SDHB; SDHA; succinate dehydrogenase; renal carcinoma
The ability to monitor the inflammatory state of the middle ear mucosa would provide clinical utility. To enable spectral measurements on the mucosa whilst rejecting background signal from the eardrum an anti-confocal system is investigated. In contrast to the central pinhole in a confocal system the anti-confocal system uses a central stop to reject light from the in-focus plane, the eardrum, with all other light detected. Monte Carlo simulations of this system show an increase in detected signal and improved signal-to-background ratio compared to a conventional confocal set-up used to image the middle ear mucosa. System parameters are varied in the simulation and their influence on the level of background rejection are presented.
(170.1790) Confocal microscopy; (170.3880) Medical and biological imaging; (170.4940) Otolaryngology; (110.0113) Imaging through turbid media
Whole-exome sequencing of two brothers with drug-resistant, early-onset, focal epilepsy secondary to extensive type IIA focal cortical dysplasia identified a paternally inherited, nonsense variant of DEPDC5 (c.C1663T, p.Arg555*). This variant has previously been reported to cause familial focal epilepsy with variable foci in patients with normal brain imaging. Immunostaining of resected brain tissue from both brothers demonstrated mammalian target of rapamycin (mTOR) activation. This report shows the histopathological features of cortical dysplasia associated with a DEPDC5 mutation, confirms mTOR dysregulation in the malformed tissue and expands the spectrum of neurological manifestations of DEPDC5 mutations to include severe phenotypes with large areas of cortical malformation.
Structured illumination microscopy (SIM) has been widely used in life science imaging applications. The maximum resolution improvement of SIM, compared to conventional bright field system is a factor of 2. Here we present an approach to structured illumination microscopy using the proximity projection grating scheme (PPGS), which has the ability to further enhance the SIM resolution without invoking any nonlinearity response from the sample. With the PPGS-based SIM, sub-100 nm resolution has been obtained experimentally, and results corresponding to 2.4 times resolution improvement are presented. Furthermore, it will be shown that an improvement of greater than 3 times can be achieved.
We describe the bi-directed eyes of a mesopelagic teleost fish, Rhynchohyalus natalensis, that possesses an extensive lateral diverticulum to each tubular eye. Each diverticulum contains a mirror that focuses light from the ventro-lateral visual field. This species can thereby visualize both downwelling sunlight and bioluminescence over a wide field of view. Modelling shows that the mirror is very likely to be capable of producing a bright, well focused image. After Dolichopteryx longipes, this is only the second description of an eye in a vertebrate having both reflective and refractive optics. Although superficially similar, the optics of the diverticular eyes of these two species of fish differ in some important respects. Firstly, the reflective crystals in the D. longipes mirror are derived from a tapetum within the retinal pigment epithelium, whereas in R. natalensis they develop from the choroidal argentea. Secondly, in D. longipes the angle of the reflective crystals varies depending on their position within the mirror, forming a Fresnel-type reflector, but in R. natalensis the crystals are orientated almost parallel to the mirror's surface and image formation is dependent on the gross morphology of the diverticular mirror. Two remarkably different developmental solutions have thus evolved in these two closely related species of opisthoproctid teleosts to extend the restricted visual field of a tubular eye and provide a well-focused image with reflective optics.
Rhynchohyalus natalensis; vision; mirror optics; deep-sea
Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.
While the need for vitamin A for the normal progression of male germ cell differentiation has been known for many years, the molecular mechanisms underlying this requirement are poorly understood. This review will explore the aspects of the effects on spermatogenesis of dietary deprivation of vitamin A, in particular as to how they compare to the male sterility that results from the genetic ablation of function of the retinoid receptor RAR·. The effects of other genes involved with retinoid synthesis, transport, and degradation are also considered. The possible cellular mechanisms that may be affected by the lack of retinoid signaling are discussed, in particular, cell cycle regulation and cell-cell interaction, both of which are critical for normal spermatogenesis.
Differentiating between malignant and benign lesions on the basis of MR images depends on the experience of the radiologist. For non-experts, we aimed to develop a simplified systematic MRI approach that uses depth, size and heterogeneity on T2 weighted MR images (T2WI) to differentiate between malignant and benign lesions, and evaluated its diagnostic accuracy.
MR images of 266 patients with histologically proven soft-tissue tumours of the extremities (102 malignant, 164 benign) were analysed according to depth (superficial or deep), size (<50, ≥50 mm) and signal intensity (homogeneous or heterogeneous) on T2WI, to determine the ability of each to predict benign and malignant tumours. These three parameters were categorised into systematic combinations of different orders of application, and each combination was assessed for its ability to differentiate between benign and malignant lesions.
Univariate analysis showed that depth, size and heterogeneity on T2WI differed significantly between benign and malignant masses (p<0.0001 each). Multiple logistic regression analysis, however, showed that depth was not helpful in distinguishing benign from malignant lesions. The systematic combination of signal intensity, size and depth, in that order, was superior to other combinations, resulting in higher diagnostic values for malignancy, with a sensitivity of 64%, a specificity of 85%, a positive predictive value of 32%, a negative predictive value of 59% and an accuracy of 77%.
A simplified systematic imaging approach, in the order signal intensity, size and depth, would be a reference to distinguish between benign and malignant soft-tissue tumours for non-experts.
Propionic acidemia (PA) is an autosomal recessive disease that results from deficiency of propionyl-CoA carboxylase (PCC). In virtually all reported cases of PA, the phenotype includes metabolic acidosis and/or neurological deficits. We report on a 14-year-old Asian male with PA who presented with isolated cardiomyopathy without any episodes of metabolic acidosis or evidence of any neurocognitive deficits. On routine metabolic screening, the patient was found to have urine organic acids suggestive of PA. Biochemical and genetic characterization confirmed a PCC deficiency with two novel mutations in PCCB: IVS7+2 T>G (c.763+2 T>G) and p.R410Q (c.1229 G>A). Residual enzyme activity likely explains our patient’s mild phenotype. Splicing mutations tend to result in a milder phenotype as these mutations may still produce small amounts of normal enzyme. In addition, the similar p.R410W mutation has been shown to have partial residual activity.
Moreover, this case illustrates the important but under-recognized manifestation of isolated cardiomyopathy as the sole clinical presentation in PA. A thorough metabolic evaluation should be performed in all pediatric patients with cardiomyopathy. Such an evaluation has important implications for clinical management and genetic counseling.
American Joint Committee on Cancer (AJCC) Tumor (T), Nodal (N) and Metastatic (M) staging is commonly used in clinical practice for treatment decisions, yet before 2004, Surveillance Epidemiology and End Results (SEER)-affiliated cancer registries did not routinely include TNM staging defined by AJCC criteria, reporting instead SEER Summary Staging.
We developed and validated an algorithm to determine AJCC TNM staging from Extent of Disease information for 17,133 female breast cancer cases diagnosed from 1988–2003 in the cancer registries of Kaiser Permanente Northern and Southern California. Test characteristics (percent agreement, Cohen’s kappa, sensitivity, specificity) were calculated to compare derived TNM with gold-standard TNM available in the registry.
Agreement for TNM variables was excellent (range 0.91–1.00 for percent agreement and Cohen’s kappa). The sensitivity and specificity, respectively, of the algorithm for AJCC TNM Version 6 staging was: Stage 0 (0.99, 1.00), Stage I (0.97, 0.98), Stage II (0.91, 0.96), Stage III (0.69, 0.99), Stage IV (0.92, 1.00). Stage III had lower sensitivity due to reclassification of supraclavicular lymph node positivity from M1 (Stage IV) to N3 (Stage IIIC) in AJCC Version 6.
Derived AJCC staging for breast tumors diagnosed before 2004 is feasible and accurate using cancer registry data.
breast cancer; American Joint Committee on Cancer; tumor; nodal; metastatic cancer staging; cancer registry; validation study
This study evaluates associations of commonly co-occurring childhood adversities with physical violence in dating relationships to identify potential strategies for refining and targeting dating violence prevention programmes.
Data on 5130 adult respondents to a nationally representative survey with at least one dating relationship before the age of 21 years were analysed. Logistic regression models assessed associations between 12 childhood adversities and physical dating violence (PDV).
Adjusting for the number of co-occurring adversities, 10 of the 12 childhood adversities were significantly associated with PDV perpetration or victimisation (OR 1.5–2.8). The population attributable risk proportion of PDV due to all 12 childhood adversities was 53.4%. Childhood adversities with the highest attributable risk proportions were sexual abuse (13.8%), interparental violence (11.6%) and parent mental illness (10.7%). Multivariate prediction equations ranked respondents by their childhood adversity risk profiles; 46.4% of PDV cases occurred in the top two risk deciles.
Assessment of a broad range of childhood exposures to familial adversities may help to identify adolescents at particularly high risk of PDV and to guide prevention efforts.
Background and Objectives
Our previous study showed protease inhibitors were attenuated by periopathogen Porphyromonas gingivalis in cultured gingival epithelial cells. We hypothesize fewer protease inhibitors would be present in more advanced periodontal sites where the level of P. gingivalis may be high. The goal of this study was to investigate the relationship between the protease inhibitor (SLPI, ELAFIN, SCCA) levels in gingival crevicular fluid (GCF) and the number of P. gingivalis in subgingival plaque.
Materials and Methods
Plaque samples from subjects without (n=18) and with moderate to advanced periodontitis (n=41) were used to quantify P. gingivalis using real-time PCR. Protease inhibitor levels in the GCF of all the subjects were determined by ELISA.
P. gingivalis was detected in 68.3% of subjects with periodontitis, while 16.7% of patients without periodontitis had detectable level of P. gingivalis. Subjects with periodontitis and P. gingivalis in their plaque exhibited lower SLPI and ELAFIN levels (p<0.001) compare to control subjects without periodontitis. SLPI was also reduced (p<0.05) in GCF of periodontal patients without detectable level of P. gingivalis. Periodontal patients with high vs. low levels of P. gingivalis exhibited reciprocal mean levels of SLPI and ELAFIN concentrations.
The reduced concentrations of SLPI and ELAFIN may contribute to the loss of host protective capacity and increase susceptibility to breakdown from chronic infection. The work of this investigation may aid in finding diagnostic and prognostic markers in periodontal health and disease and may further help in finding pharmacological targets directed against periodontal inflammation.
protease inhibitors; periodontitis; Porphyromonas gingivalis; gingival crevicular fluid
Acinar cell carcinoma of the pancreas is a rare malignant tumour developing from acinar cells, accounting for approximately 1% of pancreatic exocrine tumours. We experienced a case of an acinar cell carcinoma with fatty change. To the best of our knowledge, this is the first case report of an acinar cell carcinoma with fatty change in the clinical literature.
We examined the joint predictive effects of childhood and adolescent onset psychiatric and substance use disorders on failure to graduate high school (HS) on time. Structured diagnostic interviews were conducted with a US national sample of adults (18 and over). The analysis sample included respondents with at least 8 years of education who were born in the US or arrived in the US prior to age 13 (N=29,662). Psychiatric disorders, substance use and substance use disorders were examined as predictors of termination or interruption of educational progress prior to HS graduation, with statistical adjustment for demographic characteristics and childhood adversities. Failure to graduate HS on time was more common among respondents with any of the psychiatric and substance use disorders examined, ranging from 18.1% (specific phobia) to 33.2% (ADHD-combined type), compared with respondents with no disorder (15.2%). After adjustment for co-occurring disorders, significant associations with failure to graduate on time remained only for conduct disorder (OR=1.89, 95%CI 1.57–2.26) and the three ADHD subtypes (Inattentive OR=1.78, 95%CI 1.44–2.20, Hyperactive-Impulsive OR=1.38, 95%CI 1.14–1.67, and Combined OR=2.06, 95%CI 1.66–2.56). Adjusting for prior disorders, tobacco use was associated with failure to graduate on time (OR=1.97, 95%CI 1.80–2.16). Among substance users, substance use disorders were not associated with on time graduation. The findings suggest that the adverse impact of childhood and adolescent onset psychiatric disorders on HS graduation is largely accounted for by problems of conduct and inattention. Adjusting for these disorders, smoking remains strongly associated with failure to graduate HS on time.
Psychiatric Disorders; Educational Attainment; Epidemiology; Substance Use; Smoking
Human cathelicidin LL-37, a host defense peptide derived from leukocytes and epithelial cells, plays a crucial role in innate and adaptive immunity. Not only does it eliminate pathogenic microbes directly, LL-37 also modulates host immune responses. Emerging evidence from tumor biology studies indicates that LL-37 plays a prominent and complex role in carcinogenesis. While overexpression of LL-37 has been implicated in the development or progression of many human malignancies, including breast, ovarian and lung cancers, LL-37 suppresses tumorigenesis in gastric cancer. These data are beginning to unveil the intricate and contradictory functions of LL-37. The reasons for the tissue-specific function of LL-37 in carcinogenesis remain to be elucidated. Here, we review the relationship between LL-37, its fragments and cancer progression as well as discuss the potential therapeutic implications of targeting this peptide.
The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects.
Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients.
Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, Ktrans values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1α, IL-8, ICAM-1, and VCAM-1.
Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.
tumour microenvironment; pancreatic cancer; metronomic chemotherapy; gemcitabine; anti-angiogenesis
Gonadotrophin-releasing hormone (GnRH) neurones control the onset and maintenance of fertility. Aberrant development of the GnRH system underlies infertility in Kallmann syndrome [KS; idiopathic hypogonadotropic hypogonadism (IHH) and anosmia]. Some KS patients harbour mutations in the fibroblast growth factor receptor 1 (Fgfr1) and Fgf8 genes. The biological significance of these two genes in GnRH neuronal development was corroborated by the observation that GnRH neurones were severely reduced in newborn transgenic mice deficient in either gene. In the present study, we hypothesised that the compound deficiency of Fgf8 and its cognate receptors, Fgfr1 and Fgfr3, may lead to more deleterious effects on the GnRH system, thereby resulting in a more severe reproductive phenotype in patients harbouring these mutations. This hypothesis was tested by counting the number of GnRH neurones in adult transgenic mice with digenic heterozygous mutations in Fgfr1/Fgf8, Fgfr3/Fgf8 or Fgfr1/Fgfr3. Monogenic heterozygous mutations in Fgfr1, Fgf8 or Fgfr3 caused a 30–50% decrease in the total number of GnRH neurones. Interestingly, mice with digenic mutations in Fgfr1/Fgf8 showed a greater decrease in GnRH neurones compared to mice with a heterozygous defect in the Fgfr1 or Fgf8 alone. This compounding effect was not detected in mice with digenic heterozygous mutations in Fgfr3/Fgf8 or Fgfr1/Fgfr3. These results support the hypothesis that IHH/KS patients with digenic mutations in Fgfr1/Fgf8 may have a further reduction in the GnRH neuronal population compared to patients harbouring monogenic haploid mutations in Fgfr1 or Fgf8. Because only Fgfr1/Fgf8 compound deficiency leads to greater GnRH system defect, this also suggests that these fibroblast growth factor signalling components interact in a highly specific fashion to support GnRH neuronal development.
fibroblast growth factor receptor; fibroblast growth factor 8; gonadotrophin-releasing hormone neurones; hypogonadotropic hypogonadism; Kallmann syndrome
In spinal muscular atrophy (SMA), weakness, decreased endurance, and fatigue limit mobility. Scales have been developed to measure function across the wide spectrum of disease severity. However, these scales typically are observer dependent, and scores are based on sums across Likert-scaled items. The Six-Minute Walk Test (6MWT) is an objective, easily administered, and standardized evaluation of functional exercise capacity that has been proven reliable in other neurologic disorders and in children.
To study the performance of the 6MWT in SMA, 18 ambulatory participants were evaluated in a cross-sectional study. Clinical measures were 6MWT, 10-m walk/run, Hammersmith Functional Motor Scale–Expanded (HFMSE), forced vital capacity, and handheld dynamometry. Associations between the 6MWT total distance and other outcomes were analyzed using Spearman correlation coefficients. A paired t test was used to compare the mean distance walked in the first and sixth minutes.
The 6MWT was associated with the HFMSE score (r = 0.83, p < 0.0001), 10-m walk/run (r = −0.87, p < 0.0001), and knee flexor strength (r = 0.62, p = 0.01). Gait velocity decreased during successive minutes in nearly all participants. The average first minute distance (57.5 m) was significantly more than the sixth minute distance (48 m) (p = 0.0003).
The Six-Minute Walk Test (6MWT) can be safely performed in ambulatory patients with spinal muscular atrophy (SMA), correlates with established outcome measures, and is sensitive to fatigue-related changes. The 6MWT is a promising candidate outcome measure for clinical trials in ambulatory subjects with SMA.
= forced vital capacity;
= Hammersmith Functional Motor Scale–Expanded;
= handheld dynamometry;
= Six-Minute Walk Test;
= spinal muscular atrophy.
Nuclear factor-κB (NF-κB) is a pleiotropic transcription factor that generally enhances cellular resistance to apoptotic cell death. It has been shown to be constitutively active in some cancers and is being pursued as potential anticancer target. Sulfasalazine which is used clinically to treat Crohn's disease has emerged as a potential inhibitor of NF-κB and has shown promising results in two pre-clinical studies to target primary brain tumors, gliomas. Once digested, sulfasalazine is cleaved into sulfapyridine and 5-aminosalicylic acid (5-ASA; mesalamine) by colonic bacteria, and the latter, too, is reported to suppress NF-κB activity. We now show that glioma cells obtained from patient biopsies or glioma cell lines do not show significant constitutive NF-κB activation, unless exposed to inflammatory cytokines. This does not change when gliomas are implanted into the cerebrum of severe combined immundeficient mice. Nevertheless, sulfasalazine but not its cleaved form 5-ASA caused a dose-dependent inhibition of glioma growth. This effect was entirely attributable to the inhibition of cystine uptake via the system xc− cystine–glutamate transporter. It could be mimicked by S-4-carboxy-phenylglycine (S-4-CPG) a more specific system xc− inhibitor, and lentiviral expression of a constitutively active form of IκB kinase b was unable to overcome the growth retarding effects of sulfasalazine or S-4-CPG. Both drugs inhibited cystine uptake causing a chronic depletion of intracellular GSH and consequently compromised cellular redox defense which stymied tumor growth. This data suggests that system xc− is a promising therapeutic target in gliomas and possibly other cancers and that it can be pharmacologically inhibited by Sulfasalazine, an FDA-approved drug.
glioma; glutamate; glutathione; reactive oxygen species; S-4-carboxy-phenylglycine; system xc−
Human β-defensin-2 (hBD-2) is an antimicrobial peptide, induced by bacterial stimuli and inflammation, that plays a role in mucosal and skin innate immune defense. The nuclear factor-κB (NF-κB) transcription factor family is important in innate and adaptive immune responses to bacteria and proinflammatory cytokines. NF-κB operates via the traditional IKKβ signaling, as well as an alternative pathway utilizing IKKα signaling, which is important in keratinocyte differentiation. Our previous studies showed that pathogenic, but not commensal, bacteria used NF-κB signaling in hBD-2 induction. The objective of this study was to understand which arm of the NF-κB pathway is involved in gingival epithelial cell responses to pathogenic bacteria, including hBD-2 induction.
Cultured oral epithelial cells were transfected with synthetic small interfering RNAs (siRNAs) specific for various steps in each pathway, namely IKKβ, TRAF6 and MyD88 in the canonical, and IKKα and TRAF3 in the alternative pathway, and subsequently stimulated with various oral bacteria.
The hBD-2 induction level was reduced to 21–61% in cells in which the alternative NF-κB pathway was blocked and subsequently stimulated with pathogenic bacteria, while cells in which the canonical pathway was blocked showed reduction to 78–99%. Cells stimulated with commensals showed little change in hBD-2 induction level regardless of the siRNA used. Microarray analysis showed that oral epithelia differentially regulated numerous innate immune markers in response to pathogens and commensals.
Our data suggest a role for the IKKα/TRAF3 pathway in NF-κB activation by pathogenic bacteria, while commensal bacteria do not utilize either NF-κB pathway, for hBD-2 induction.
commensals; gingival epithelium; innate immunity; nuclear factor-κB; pathogens
Growth arrest-specific gene 6 (Gas6), identified in 1995, acts as the ligand to the Axl/Tyro3 family of tyrosine kinase receptors and exerts mitogenic activity when bound to these receptors. Overexpression of the Axl/Tyro3 receptor family has been found in breast, ovarian and lung tumours. Gas6 is upregulated 23-fold by progesterone acting through the progesterone receptor B (PRB). Recently, Gas6 has been shown to be a target for overexpression and amplification in breast cancer. Quantitative real-time PCR analysis was used to determine the levels of Gas6 mRNA expression in 49 primary breast carcinomas. Expression of PRB protein was evaluated immunohistochemically with a commercially available PRB antibody. The results showed a positive association between PRB protein and Gas6 mRNA levels (P=0.04). Gas6 correlated positively with a number of favourable prognostic variables including lymph node negativity (P=0.0002), younger age at diagnosis (P=0.04), smaller size of tumours (P=0.02), low Nottingham prognostic index scores (P=0.03) and low nuclear morphology (P=0.03). This study verifies for the first time the association between PRB and Gas6 in breast cancer tissue.
growth arrest-specific gene 6; Axl tyrosine kinase receptor; progesterone receptor B; breast cancer
Malignant gliomas have been shown to release glutamate, which kills surrounding brain cells, creating room for tumor expansion. This glutamate release occurs primarily via system
xC−, a Na+-independent cystine-glutamate exchanger. We show here, in addition, that the released glutamate acts as an essential autocrine/paracrine signal that promotes cell invasion. Specifically, chemotactic invasion and scrape motility assays each show dose-dependent inhibition of cell migration when glutamate release was inhibited using either S-(4)-CPG or sulfasalazine, both potent blockers of system
xC−. This inhibition could be overcome by the addition of exogenous glutamate (100 μmol/L) in the continued presence of the inhibitors. Migration/invasion was also inhibited when Ca2+-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPA-R) were blocked using GYKI or Joro spider toxin, whereas CNQX was ineffective. Ca2+ imaging experiments show that the released glutamate activates Ca2+-permeable AMPA-R and induces intracellular Ca2+ oscillations that are essential for cell migration. Importantly, glioma cells release glutamate in sufficient quantities to activate AMPA-Rs on themselves or neighboring cells, thus acting in an autocrine and/or paracrine fashion. System
xC− and the appropriate AMPA-R subunits are expressed in all glioma cell lines, patient-derived glioma cells, and acute patient biopsies investigated. Furthermore, animal studies in which human gliomas were xenographed into scid mice show that chronic inhibition of system
xC−–mediated glutamate release leads to smaller and less invasive tumors compared with saline-treated controls. These data suggest that glioma invasion is effectively disrupted by inhibiting an autocrine glutamate signaling loop with a clinically approved candidate drug, sulfasalazine, already in hand.
Background and purpose:
Thoracic aortic dissection is a life-threatening complication of Marfan syndrome, a connective tissue disorder caused by mutations in the gene encoding fibrillin-1. We have demonstrated that nitric oxide-mediated endothelial-dependent relaxation is impaired in the thoracic aorta in Marfan syndrome. In the present study, we determined whether the cyclooxygenase (COX)-pathway is involved in the compromised aortic vasomotor function.
Thoracic aortae from mice at 3, 6 and 9 months of age, heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1
C1039G/+, ‘Marfan', n=35), were compared with those from age-matched controls (n=35).
Isometric force measurement revealed that preincubation with indomethacin, a non-specific COX inhibitor, but not valeryl salicylate, a specific COX-1 inhibitor, improved the phenylephrine-induced contractions (at 6 months, EC50 and Emax were increased 4.5-fold and by 45%, respectively) in Marfan aortae. Sensitivity to acetylcholine-induced relaxation was improved 10-fold. Blockade of the thromboxane-endoperoxide receptor by SQ-29548 did not affect phenylephrine-mediated contractions in Marfan aortae, although they did respond to the thromboxane analogue, U46619. From 6 months on, phenylephrine-induced secretion of prostacyclin and thromboxane A2 in Marfan aortae was 200% and 40%, respectively, of those in controls. Reduced COX-1 expression was detected in Marfan aortae at 3 and 9 months, whilst COX-2 expression was increased from 3 months on.
Conclusions and implications:
The compromised vasomotor function in Marfan thoracic aortae is associated with an imbalanced synthesis of thromboxane A2 and prostacyclin resulting from the differential protein expression of COX-1 and COX-2.
Marfan syndrome; cyclooxygenase; vasoconstriction; vasorelaxation; endothelial function; thoracic aorta; age-dependent disease progression; thromboxane A2; prostacyclin; acetylcholine