Endothelial TNF receptor 2 induces IRF1 transcription factor-dependent interferon-ß autocrine signaling to promote monocyte recruitment.

Endothelial-dependent mechanisms of mononuclear cell influx are not well understood. We showed that acute stimulation of murine microvascular endothelial cells expressing the tumor necrosis factor receptors TNFR1 and TNFR2 with the soluble cytokine TNF led to CXCR3 chemokine generation. The TNF receptors signaled through interferon regulatory factor-1 (IRF1) to induce interferon-ß (IFN-ß) and subsequent autocrine signaling via the type I IFN receptor and the transcription factor STAT1. Both TNFR2 and TNFR1 were required for IRF1-IFNß signaling and, in human endothelial cells TNFR2 expression alone induced IFN-ß signaling and monocyte recruitment. In vivo, TNFR1 was required for acute renal neutrophil and monocyte influx after systemic TNF treatment, whereas the TNFR2-IRF1-IFN-ß autocrine loop was essential only for macrophage accumulation. In a chronic model of proliferative nephritis, IRF1 and renal-expressed TNFR2 were essential for sustained macrophage accumulation. Thus, our data identify a pathway in endothelial cells that selectively recruits monocytes during a TNF-induced inflammatory response.

GAPO Syndrome-A Rare Cause of Osteomyelitis of Jaws; Report of 4 Cases With a Brief Review of the Literature.

GAPO syndrome is characterized by growth retardation, alopecia, pseudoanodontia, and ophthalmic abnormalities. This very rarely reported syndrome affects various ethnic groups and can present with manifestations other than those usually reported. Pseudoanodontia is a rare clinical and radiologic manifestation that is always associated with GAPO syndrome. Osteomyelitis of the jaws is a less common disease that is usually attributed to odontogenic causes. This case report describes osteomyelitis of the mandible in a patient with GAPO syndrome. Further, an additional 3 cases of GAPO in the patient's family, with special emphasis on oral mucosal changes and pseudoanodontia, are discussed.

AKAP9, a Regulator of Microtubule Dynamics, Contributes to Blood-Testis Barrier Function.

The blood-testis barrier (BTB), formed between adjacent Sertoli cells, undergoes extensive remodeling to facilitate the transport of preleptotene spermatocytes across the barrier from the basal to apical compartments of the seminiferous tubules for further development and maturation into spermatozoa. The actin cytoskeleton serves unique structural and supporting roles in this process, but little is known about the role of microtubules and their regulators during BTB restructuring. The large isoform of the cAMP-responsive scaffold protein AKAP9 regulates microtubule dynamics and nucleation at the Golgi. We found that conditional deletion of Akap9 in mice after the initial formation of the BTB at puberty leads to infertility. Akap9 deletion results in marked alterations in the organization of microtubules in Sertoli cells and a loss of barrier integrity despite a relatively intact, albeit more apically localized F-actin and BTB tight junctional proteins. These changes are accompanied by a loss of haploid spermatids due to impeded meiosis. The barrier, however, progressively reseals in older Akap9 null mice, which correlates with a reduction in germ cell apoptosis and a greater incidence of meiosis. However, spermiogenesis remains defective, suggesting additional roles for AKAP9 in this process. Together, our data suggest that AKAP9 and, by inference, the regulation of the microtubule network are critical for BTB function and subsequent germ cell development during spermatogenesis.

RhoA-mediated signaling in Notch-induced senescence-like growth arrest and endothelial barrier dysfunction.

OBJECTIVE: Notch signaling has a critical role in vascular development and morphogenesis. Activation of Notch in endothelial cells led to a senescence-like phenotype with loss of barrier function. Our objective was to understand the molecular pathways mediating this phenotype. METHODS AND RESULTS: Human primary endothelial cells increase expression of Notch receptors and ligands during propagation in vitro toward natural senescence. This senescence was induced at low passage with Notch activation. We characterized the pathways activated downstream of Notch signaling. Notch was activated by Delta-like 4 ligand or constitutively active Notch receptors and measured for cell proliferation, migration, and sprouting. Notch signaling triggered early senescence in low-passage cells, characterized by increased p53 and p21 expression. The senescence phenotype was associated with hyperpermeability of the monolayer, with disrupted vascular endothelial cadherin and ß-catenin levels and localization. Consistent with changes in cell shape and contact, we demonstrated that Notch activation increases myosin light chain phosphorylation by activating Rho kinase. Inhibition of Rho abrogated Notch-induced myosin light chain phosphorylation and led to enhanced barrier function by reorganizing F-actin to ß-catenin-containing cell-cell adherens junctions. CONCLUSIONS: Our findings show that RhoA/Rho kinase regulation by Notch signaling in endothelial cells triggers a senescence phenotype associated with endothelial barrier dysfunction.

Notch and transforming growth factor-beta (TGFbeta) signaling pathways cooperatively regulate vascular smooth muscle cell differentiation.

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation.

Development of bone and adipose tissue are linked processes arising from a common progenitor cell, but having an inverse relationship in disease conditions such as osteoporosis. Cellular differentiation of both tissues relies on growth factor cues, and we focus this study on Sprouty1 (Spry1), an inhibitor of growth factor signaling. We tested whether Spry1 can modify the development of fat cells through its activity in regulating growth factors known to be important for adipogenesis. We utilized conditional expression and genetic-null mouse models of Spry1 in adipocytes using the fatty acid binding promoter (aP2). Conditional deletion of Spry1 results in 10% increased body fat and decreased bone mass. This phenotype was rescued on Spry1 expression, which results in decreased body fat and increased bone mass. Ex vivo bone marrow experiments indicate Spry1 in bone marrow and adipose progenitor cells favors differentiation of osteoblasts at the expense of adipocytes by suppressing CEBP-beta and PPARgamma while up regulating TAZ. Age and gender-matched littermates expressing only Cre recombinase were used as controls. Spry1 is a critical regulator of adipocyte differentiation and mesenchymal stem cell (MSC) lineage allocation, potentially acting through regulation of CEBP-beta and TAZ.

Cardiovascular and hematopoietic defects associated with Notch1 activation in embryonic Tie2-expressing populations.

Notch signaling is critical for the development and maintenance of the cardiovasculature, with loss-of-function studies defining roles of Notch1 in the endothelial/hematopoietic lineages. No in vivo studies have addressed complementary gain-of-function strategies within these tissues to define consequences of Notch activation. We developed a transgenic model of Cre recombinase-mediated activation of a constitutively active mouse Notch1 allele (N1ICD(+)) and studied transgene activation in Tie2-expressing lineages. The in vivo phenotype was compared to effects of Notch1 activation on endothelial tubulogenesis, paracrine regulation of smooth muscle cell proliferation, and hematopoiesis. N1ICD(+) embryos showed midgestation lethality with defects in angiogenic remodeling of embryonic and yolk sac vasculature, cardiac development, smooth muscle cell investment of vessels, and hematopoietic differentiation. Angiogenic defects corresponded with impaired endothelial tubulogenesis in vitro following Notch1 activation and paracrine inhibition of smooth muscle cells when grown with Notch1-activated endothelial cells. Flow cytometric analysis of hematopoietic and endothelial precursor populations demonstrated a significant loss of CD71(+)/Ter119(+) populations with an active N1ICD(+) allele and a corresponding increase in c-Kit(+)/CD71 and Flk1(+) populations, suggesting a developmental block during the transition between c-Kit- and Ter119-expressing erythroblasts. Cardiovascular lineages are sensitive to an imbalance in Notch signaling, with aberrant activation reflecting a vascular phenotype comparable to a loss-of-function Notch1 mutation.

Frequency and variability of five non metric dental crown traits in the permanent maxillary dentitions of a racially mixed population from Bengaluru, Karnataka.

INTRODUCTION: Evaluation of Tooth Crown non metric traits benefits to assess the biological distance between populations. It is well known that these traits are characterised by a high inter-population differentiation, low sexual dimorphism, and their recording is loaded by relatively small intra and inter observer error. The dental morphological traits are successfully used in the description and explanation of the microevolutionary and ethnogenetic processes. This paper presents the results of the permanent maxillary dentition tooth crown traits differentiation of human populations from Bengaluru. MATERIALS AND METHODS: The comparative analysis was carried out on the basis of 4 groups for 5 Tooth Crown non metric traits in maxillary permanent dentition using Arizona State University Dental Anthropology System for morphological scoring system of each trait and each score was charted on Osteoware Dental Morphology software. RESULTS: Study analysed 400 dental casts from 4 different ethnic groups. Traits Winging, shovelling, lingual tubercle showed highest expression in Iranians while Cusp of Carabelli's trait expression showed 87% of prevalence in the surveyed group but the Hypocone trait showed the highest expression in Muslims. CONCLUSION: Traits of the human dentition can be a valuable diagnostic tool for anthropological studies in classifying and characterizing different ethnic groups. According to the results obtained from this study, it can be said that the groups Hindus, Muslims and Christians (Indians) belong to Sundonts, while Iranians fall under sinodonts population group.

Mucormycosis in immunocompetent patient resulting in extensive maxillary sequestration.

Mucormycosis or zygomycosis, also called phycomycosis, is an uncommon, invasive, potentially lethal and an aggressive fungal infection of the order Mucorales that usually affects patients with alteration of their immunological system. From its initial description (Paltauf, 1885), this entity still has a high mortality. Imaging techniques are not usually diagnostic, and cultures are not totally reliable. Definitive diagnosis is exclusively obtained by means of histopathological examination. Early recognition and aggressive treatment are of paramount importance and have reduced the mortality and morbidity. We present here a case report of oral mucormycosis in a 32-year-old male, immunocompetent individual resulting in extensive maxillary sequestration.

Study of salivary arecoline in areca nut chewers.

AIMS: Arecoline, a predominant alkaloid present in arecanut, has been implicated in the pathogenesis of several oral diseases because of its mutagenic and carcinogenic potential. The response of cultured cells to arecoline is highly dependent on its concentration; arecoline stimulates cultured cells above 0.1 µg/ml and is cytotoxic above 10 µg/ ml. Although this alkaloid seems important for areca nut induced oral diseases and carcinogenesis, little is known of the levels achieved before, during and after chewing. Also, it is prudent to understand its effects in arecanut chewers for a comprehensive understanding of its pathogenesis. Accordingly, the present study quantified the salivary arecoline levels in arecanut chewers. MATERIALS AND METHODS: The study participants were divided into Study Group A & B and Control Group C; unstimulated whole saliva was collected by spitting method for a period of 5 min. Then, participants in Group A and C chewed 0.5 g of areca nut without any other additives while in Group B were asked to chew 0.5 g of inert rubber base impression material. Stimulated whole saliva from all three groups was collected into graduated tubes during chewing at time intervals of 1, 3, 5, 10, 15, 20 and 25 min. Then, all participants were asked to remove nut particles or inert rubber base material from the mouth, and saliva samples were collected further up to 20 min, changing tubes at 5 min interval. Salivary arecoline was quantitated by HPLC-MS. The tabulation and descriptive statistics of the study were carried out. RESULTS: In the present study, baseline levels of arecoline were zero in all three groups, whereas mean salivary arecoline levels during chewing were 76.93 ng/ml, 129.83 ng/ml and 64.83 ng/ml and after chewing were 196.17 ng/ml, 321.12 ng/ml and 43.75 ng/ml in Groups A, B and Control respectively, which were significantly higher than reported threshold levels. CONCLUSIONS: The data from this study reveals that a significant amount of arecoline would be trapped in oral cavity, or being re-circulated between blood and saliva might have resulted in surprisingly high levels of arecoline even 10 mins after chewing in both groups after which the levels started declining. The higher levels of salivary arecoline achieved during and after chewing are enough to cause cytotoxic and genotoxic effects on oral tissues over a period of time in chronic chewers. The great differences in salivary arecoline levels achieved during chewing, may contribute to the variable response to areca nut seen in communities where this habit is widespread. Areca nut users have persistent background salivary arecoline levels long after chewing, whereas concentrations achieved are highly variable and consistent with a role in oral pre-malignancy and malignancy..

The cusp of Carabelli: Frequency, distribution and type in the Bengaluru population.

AIMS: Detailed description and study of teeth traits could provide valuable information regarding phylogeny of man and distinctions between races and subraces. But morphological variations of the human dentition have not been utilized to their full potential by anthropologists concerned with patterns of human biological variation in Indian population. The aim of the present study is to detect the frequency and degree of expression of Carabelli's trait in Bengaluru population, this helps to develop a probabilistic model to distinguish individuals from specific human populations, particularly for forensic purposes. MATERIALS AND METHODS: A total number 400of age and sex matched individuals from four different ethnic groups - Hindu, Islam, Christian and Iranians were examined clinically and study casts were made. Permanent maxillary first molars were examined for the expression of Carabelli's trait, Dahlberg classification system was used to score the trait on the teeth. The scores were recorded on Osteoware Dental Morphology software. The cast were examined by 2 observers independently to eliminate intra observer variation in interpretation and mean of 2 was taken for analysis. The data so obtained was statistically analysed especially emphasizing on differences between above mentioned 4 ethnic groups. RESULTS: Cusp of Carabelli was present in 87% of the study population in maxillary first permanent molar. Type 3 was the most frequently expressed and Type 6 was the least frequently expressed and both type being expressed in Islamic groups. The expression of trait was bilateral in 90% of the surveyed groups. CONCLUSIONS: It was concluded that the prevalence of cusp of Carabelli in the small population from Bengaluru considered in the present study was found to possess a high degree of Carabelli trait expression.

Gingival mesenchymal stem cells.

The human gingiva, characterized by its outstanding scarless wound healing properties, is a unique tissue and a pivotal component of the periodontal apparatus, investing and surrounding the teeth in their sockets in the alveolar bone. In the last year's gingival mesenchymal stem/progenitor cells (GMSCs), with promising regenerative and immunomodulatory properties, have been isolated and characterized from the gingival lamina propria. These cells, in contrast to other mesenchymal stem/progenitor cell (MSC) sources, are abundant, readily accessible and easily obtainable through minimally invasive cell isolation techniques. This short communication summarizes the current scientific evidence on GMSCs.

The ß-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance.

Resistance to fungal infections is attributed to engagement of host pattern-recognition receptors, notably the ß-glucan receptor Dectin-1 and the integrin Mac-1, which induce phagocytosis and antifungal immunity. However, the mechanisms by which these receptors coordinate fungal clearance are unknown. We show that upon ligand binding, Dectin-1 activates Mac-1 to also recognize fungal components, and this stepwise process is critical for neutrophil cytotoxic responses. Both Mac-1 activation and Dectin-1- and Mac-1-induced neutrophil effector functions require Vav1 and Vav3, exchange factors for RhoGTPases. Mac-1- or Vav1,3-deficient mice have increased susceptibility to systemic candidiasis that is not due to impaired neutrophil recruitment but defective intracellular killing of C. albicans yeast forms, and Mac-1 or Vav1,3 reconstitution in hematopoietic cells restores resistance. Our results demonstrate that antifungal immunity depends on Dectin-1-induced activation of Mac-1 functions that is coordinated by Vav proteins, a pathway that may localize cytotoxic responses of circulating neutrophils to infected tissues.

Molecular mechanisms of anti-angiogenic effect of curcumin.

Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 microM-1mM) for various time periods (0-24h) confirm its angioinhibitory action at the level of gene expression. Because of its non-toxic nature, curcumin could be further developed to treat chronic diseases that are associated with extensive neovascularization.