Evaluation of the Effectiveness of Amlexanox in the Treatment of Erosive Oral Lichen Planus: A Clinical Experience from a Tertiary Care Center.

Aim: This comparative study evaluated the effectiveness and safety profile of topical amlexanox and triamcinolone for the management of erosive oral lichen planus (EOLP). Materials and Methods: This prospective, observational study included 21 patients diagnosed clinically and histopathologically with EOLP and categorized into two groups. Subjects in the two groups were prescribed topical amlexanox and triamcinolone, respectively, for 4 weeks. The area of the erosive lesion and burning sensation was measured at baseline, at the end of the first, 2second, and fourth week. These outcome measures were documented and statistically analyzed. The statistical analyses were performed using the IBM SPSS Statistics version 22. Analysis for age distribution was done by independent sample t test. Analysis of sex distribution was done by chi-square test. Variations within a single group for both the outcome parameters were calculated by Wilcoxon signed rank test. (P < 0.05 statistically significant). Results: A total of 30 erosive sites were evaluated in 21 patients over a 4-week duration. The most common site was the buccal mucosa in both groups (23 of 30; 76.67% of total lesions assessed), followed by the tongue (5 of 30; 16.67% of total lesions assessed), the palate (1 of 30; 3.33% of total sites assessed), and the maxillary attached gingiva (1 of 30; 3.33% of total sites assessed). Group 1 (amlexanox) was comprised of 11 subjects, whereas Group 2 (triamcinolone) was comprised of 10 subjects. Pre and posttreatment comparison revealed no statistically significant difference (P = 0.756; 0.512, respectively), for the area of the erosion and burning sensation. Intragroup analysis showed that in Groups 1 and 2, there was a statistically significant reduction in the measures posttreatment (P < 0.05). Conclusions: Amlexanox provides an earlier onset of pain relief in the treatment of EOLP, whereas providing a comparable reduction in the erosive area compared with triamcinolone. Topical amlexanox appears to be as effective as triamcinolone and is a promising alternative in the management of the erosive lichen planus with minimal adverse effects.

Dosimetric impact of dwell time deviation constraint on inverse brachytherapy treatment planning and comparison with conventional optimization method for interstitial brachytherapy implants.

PURPOSE: High-dose rate remote afterloading brachytherapy machine and advanced treatment planning system help in getting optimum dose to tumor and low dose to normal structures. Inverse planning simulated annealing (IPSA) optimization technique has a unique feature of dwell time deviation constraint (DTDC). In this study, six IPSA-based plans having different DTDC values with routinely practiced geometric plus graphical optimization (GO + GrO) have been compared using various dosimetric parameters. MATERIALS AND METHODS: For this retrospective study, we have generated IPSA-optimized interstitial brachytherapy plans for ten cancer cervix patients. Routinely practiced GO + GrO-based plans were compared with six different IPSA plans having varying DTDC values from 0.0 to 1.0 using different dosimetric indices. RESULTS: Conformity index and homogeneity index (HI) were higher in GO + GrO plans, compared to IPSA-optimized plans. However, HI of IPSA plans was increasing with increasing DTDC values. High-dose volumes were well controllable using DTDC parameter in IPSA-optimized plans. Dose to the rectum and bladder was smaller for IPSA-optimized plans than GO + GrO plans. CONCLUSIONS: One of the benefits of applying DTDC in IPSA-optimized plan is that it reduces high-dose volumes. Another advantage is the reduction in rectum and bladder dose.

Evaluation of candidal species among individuals with oral potentially malignant disorders and oral squamous cell carcinoma.

CONTEXT: Cancer afflicts almost all communities worldwide. Although it arises de novo in many instances, a significant proportion of oral squamous cell carcinoma (OSCC) develops from potentially malignant disorders (PMDs). Further, the association of Candida with various potentially malignant and malignant lesions has been reported as a causative agent. AIMS: The aim of the study is to evaluate and intercompare the predominant candidal species among individuals with PMD and OSCC. SUBJECTS AND METHODS: The swab samples were collected for the microbiological culture followed by incisional biopsy for histopathological confirmation. The swab samples were streaked and incubated on Sabouraud-dextrose agar medium and positive candidal colonies were incubated on CHROM agar for speciation. SETTINGS AND DESIGN: A total of clinically diagnosed 95 subjects of which 25 as normal controls, 30 as PMDs and 40 as OSCC were included. The collected swab samples were initially streaked and incubated on Sabouraud dextrose agar (SDA) medium, and later, only positive candidal colonies were incubated on CHROM agar for speciation. STATISTICAL ANALYSIS: Chi-square test was utilized. RESULTS: Positive candidal growth on SDA medium was seen in 24%, 43% and 82% and negative in 76%, 57% and 18% individuals of normal controls, PMDs and OSCC, respectively. On evaluation on Chromagar medium, Candida species was present in 20%, 40% and 77% and absent in 80%, 60% and 23% individuals among controls, PMDs and OSCC group, respectively. On speciation of Candida in CHROMagar among the controls, PMDs and OSCC, Candida albicans species was present in 4 (16%), 7 (23%) and 4 (10%); Candida krusei in 1 (4%), 5 (17%) and 10 (25%); Candida glabrata in nil, nil and 6 (20%) and Candida tropicalis in nil, nil, and 2 (5%) cases, respectively. CONCLUSION: There was predominant carriage of candidal species in PMDs and OSCC, but whether Candida has specific establishment in PMDs or in malignancy is still a matter of debate.

Study of the effects of dwell time deviation constraints on inverse planning simulated annealing optimized plans of intracavitary brachytherapy of cancer cervix.

PURPOSE: High Dose Rate (HDR) remote afterloading brachytherapy machine and advanced treatment planning system have made it possible to make variations in individual dwell times across a catheter according to tumour density and for sparing normal structures. New inverse planning technique such as Inverse Planning Simulated Annealing (IPSA) has also been introduced. But very few institutions are venturing towards volume based IPSA optimised intracavitary brachytherapy. This study focuses on dwell time deviation constraint (DTDC) feature of IPSA based optimization which restricts the large variation of dwell time across the catheter. METHODS AND MATERIAL: For this retrospective study we have generated IPSA optimised intracavitary brachytherapy plans for 20 cancer cervix applications. The initial DTDC value of each IPSA plan was kept 0.0. Later on gradual increment was made in DTDC values in step of 0.2. Plan modulation index (M) defined by Ryan L. Smith et al was used for characterising the variation of dwell time modulation with respect to gradual increase in DTDC parameter. RESULTS: Plan modulation index gradually decreases with increasing value of DTDC from 0.0 to 1.0. There was the 83% decrease in M value from IPSA of DTDC 0.0 to fully constrained IPSA of DTDC1.0. There is reduction of 8.26% and 6.95% for D2cc values of rectum and bladder respectively for DTDC 1.0 compared to DTDC 0.0. CONCLUSIONS: One of the benefits of applying DTDC constrained in IPSA plan is that, it removes local hot spots. It's another advantage is the reduction in rectum and bladder dose.

Glandular Odontogenic Cyst in Maxilla: A Case Series.

Glandular odontogenic cyst is rare phenomenon with 0.012% to 0.03% frequency of all jaw cysts and worldwide prevalence of 0.17%. Diagnosis of Glandular odontogenic cyst, well known for its aggressive growth potential and high rate of recurrence, is very crucial. This report presents cases of two 50-year old individuals with Glandular odontogenic cyst presenting as a radiolucent lesion of maxilla. Final diagnosis was made on the basis of histopathological features and further confirmed by immunohistochemical analysis. Keywords: histology; immunohistochemistry; odontogenic cyst.

Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.

Targeting cancer metabolism by simultaneously disrupting parallel nutrient access pathways.

Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity.

Azacyclic FTY720 Analogues That Limit Nutrient Transporter Expression but Lack S1P Receptor Activity and Negative Chronotropic Effects Offer a Novel and Effective Strategy to Kill Cancer Cells in Vivo.

FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.

Nitric Oxide Synthase as a Target for Methicillin-Resistant Staphylococcus aureus.

Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme.

Reciprocal effects of rab7 deletion in activated and neglected T cells.

Mouse models lacking proteins essential for autophagosome formation have demonstrated that autophagy plays a critical role in T cell development and activation. To better understand the function of autophagy in quiescent and activated lymphocytes, we have generated a mouse deficient in rab7 selectively in T cells and compared the effects of blocking autophagy at an early (atg5(-/-)) or late (rab7(-/-)) stage on T cell biology. rab7(-/-) murine embryonic fibroblasts (MEFs) and T cells generated from these mice exhibit a profound block in autophagosome degradation and are as sensitive as atg5(-/-) cells to extracellular nutrient limitation. Rab7(flox/flox)CD4-Cre(+) mice lacking the RAB7 protein in both CD4 and CD8 T cells had reduced numbers of peripheral T cells, but this defect was not as severe as in Atg5(flox/flox)CD4-Cre(+) mice despite efficient rab7 deletion and inhibition of autophagic flux. This difference may stem from the reduced ROS generation and enhanced survival of rab7(-/-) T cells compared with wild-type and atg5(-/-) T cells in the absence of cytokine stimulation. rab7(-/-) and atg5(-/-) T cells exhibited similar defects in proliferation both following antibody-mediated T cell receptor (TCR) cross-linking and using a more physiologic activation protocol, allogeneic stimulation. Interestingly, autophagy was not required to provide building blocks for the upregulation of nutrient transporter proteins immediately following activation. Together, these studies suggest that autophagosome degradation is required for the survival of activated T cells, but that loss of rab7 is better tolerated in naïve T cells than the loss of atg5.

Nutritional and hormonal regulation of the TOR effector 4E-binding protein (4E-BP) in the mosquito Aedes aegypti.

Mosquitoes require blood for egg development, and, as a consequence, they transmit pathogens of devastating diseases. Target of rapamycin (TOR) signaling is a key pathway linking blood feeding and egg development in the mosquito Aedes aegypti. We show that the regulation of the TOR effector translational repressor 4E-BP is finely tuned to the nutritional requirements of the female mosquito, and it occurs at transcriptional and post-translational levels. Immediately after blood feeding, 4E-BP became hyperphosphorylated, suggesting rapid inhibition of its translational repression function. 4E-BP was highly phosphorylated after in vitro incubation of the fat body in the presence of amino acids; this phosphorylation was rapamycin insensitive, in contrast to another TOR target, S6K, phosphorylation of which was rapamycin sensitive. A high level of 4E-BP phosphorylation was also elicited by insulin. Rapamycin and the PI3K inhibitor LY294002 blocked insulin-mediated 4E-BP phosphorylation. RNA-interference depletion of the insulin receptor or Akt resulted in severe reduction of 4E-BP phosphorylation. Phosphorylation and stability of 4E-BP was dependent on its partner eIF4E translation initiation factor. Silencing of 4E-BP resulted in reduction of the life span of adult female mosquitoes. This study demonstrates a dual nutritional and hormonal control of 4E-BP and its role in mosquito egg development.

PTEN negatively regulates engulfment of apoptotic cells by modulating activation of Rac GTPase.

Efficient clearance of apoptotic cells by phagocytes (efferocytosis) is critical for normal tissue homeostasis and regulation of the immune system. Apoptotic cells are recognized by a vast repertoire of receptors on macrophage that lead to transient formation of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and subsequent cytoskeletal reorganization necessary for engulfment. Certain PI3K isoforms are required for engulfment of apoptotic cells, but relatively little is known about the role of lipid phosphatases in this process. In this study, we report that the activity of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatidylinositol 3-phosphatase, is elevated upon efferocytosis. Depletion of PTEN in macrophage results in elevated PtdIns(3,4,5)P(3) production and enhanced phagocytic ability both in vivo and in vitro, whereas overexpression of wild-type PTEN abrogates this process. Loss of PTEN in macrophage leads to activation of the pleckstrin homology domain-containing guanine-nucleotide exchange factor Vav1 and subsequent activation of Rac1 GTPase, resulting in increased amounts of F-actin upon engulfment of apoptotic cells. PTEN disruption also leads to increased production of anti-inflammatory cytokine IL-10 and decreased production of proinflammatory IL-6 and TNF-α upon engulfment of apoptotic cells. These data suggest that PTEN exerts control over efferocytosis potentially by regulating PtdIns(3,4,5)P(3) levels that modulate Rac GTPase and F-actin reorganization through Vav1 exchange factor and enhancing apoptotic cell-induced anti-inflammatory response.

Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling.

Inositol phosphates are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here we demonstrate that disruption of inositol hexakisphosphate kinase 1 (InsP6K1), one of the three mammalian inositol hexakisphosphate kinases (InsP6Ks) that convert inositol hexakisphosphate (InsP6) to InsP7, conferred enhanced phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3)-mediated membrane translocation of the pleckstrin homology domain of the kinase Akt and thus augmented downstream PtdIns(3,4,5)P3 signaling in mouse neutrophils. Consequently, these neutrophils had greater phagocytic and bactericidal ability and amplified NADPH oxidase-mediated production of superoxide. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. In contrast, an increase in intracellular InsP7 blocked chemoattractant-elicited translocation of the pleckstrin homology domain to the membrane and substantially suppressed PtdIns(3,4,5)P3-mediated cellular events in neutrophils. Our findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

Pretreatment with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670 augments the efficacy of granulocyte transfusion in a clinically relevant mouse model.

The clinical outcome of granulocyte transfusion therapy is often hampered by short ex vivo shelf life, inefficiency of recruitment to sites of inflammation, and poor pathogen-killing capability of transplanted neutrophils. Here, using a recently developed mouse granulocyte transfusion model, we revealed that the efficacy of granulocyte transfusion can be significantly increased by elevating intracellular phosphatidylinositol (3,4,5)-trisphosphate signaling with a specific phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670. Neutrophils treated with SF1670 were much sensitive to chemoattractant stimulation. Neutrophil functions, such as phagocytosis, oxidative burst, polarization, and chemotaxis, were augmented after SF1670 treatment. The recruitment of SF1670-pretreated transfused neutrophils to the inflamed peritoneal cavity and lungs was significantly elevated. In addition, transfusion with SF1670-treated neutrophils led to augmented bacteria-killing capability (decreased bacterial burden) in neutropenic recipient mice in both peritonitis and bacterial pneumonia. Consequently, this alleviated the severity of and decreased the mortality of neutropenia-related pneumonia. Together, these observations demonstrate that the innate immune responses can be enhanced and the severity of neutropenia-related infection can be alleviated by augmenting phosphatidylinositol (3,4,5)-trisphosphate in transfused neutrophils with PTEN inhibitor SF1670, providing a therapeutic strategy for improving the efficacy of granulocyte transfusion.

The small GTPase Rheb is a key component linking amino acid signaling and TOR in the nutritional pathway that controls mosquito egg development.

Mosquitoes transmit numerous devastating human diseases because they require blood feeding for egg development. Previously, we have shown that the nutritional Target-of-Rapamycin (TOR) pathway mediates blood-meal activation of mosquito reproductive cycles. Blood-derived amino acid (AA) signaling through the nutrient-sensitive TOR kinase is critical for the transcriptional activation of the major yolk protein precursor (YPP) gene, vitellogenin (Vg), initiation of vitellogenesis and egg development. In this study, we provide in vitro and in vivo evidence that the Rheb GTPase (Ras Homologue Enriched in Brain), which is an upstream activator of TOR, is required for AA-mediated activation of the TOR pathway in the fat body of the mosquito Aedes aegypti. Using RNA interference (RNAi) methods, we showed that Rheb was indispensable in AA-induced phosphorylation of S6 kinase, a key downstream substrate of TOR activation. Rheb RNAi depletion resulted in significant downregulation of Vg transcription and translation in the mosquito fat body, which was monitored in vivo after blood meal or in vitro organ culture after AA stimulation. Egg development was severely hindered in mosquitoes with a Rheb RNAi depletion background. This study represents a notable step in deciphering molecular pathways controlling reproduction of this important vector of human diseases.

Effect of insulin and 20-hydroxyecdysone in the fat body of the yellow fever mosquito, Aedes aegypti.

In mosquitoes, yolk protein precursor (YPP) gene expression is activated after a blood meal through the synergistic action of a steroid hormone and the amino acid/target of rapamycin (TOR) signaling pathway in the fat body. We investigated the role of insulin signaling in the regulation of YPP gene expression. The presence of mosquito insulin receptor (InR) and the Protein kinase B (PKB/Akt) in the adult fat body of female mosquitoes was confirmed by means of the RNA interference (RNAi). Fat bodies stimulated with insulin were able to promote the phosphorylation of ribosomal S6 Kinase, a key protein of the TOR signaling pathway. Importantly, insulin in combination with 20-hydroxyecdysone activated transcription of the YPP gene vitellogenin (Vg), and this process was sensitive to the phosphoinositide-3 kinase (PI-3k) inhibitor LY294002 as well as the TOR inhibitor rapamycin. RNAi-mediated knockdown of the mosquito InR, Akt, and TOR inhibited insulin-induced Vg gene expression as well as S6 Kinase phosphorylation in in vitro fat body culture assays.

Target of rapamycin-dependent activation of S6 kinase is a central step in the transduction of nutritional signals during egg development in a mosquito.

Female mosquitoes are effective disease vectors, because they take blood from vertebrate hosts to obtain nutrients for egg development. Amino acid signaling via the target of rapamycin (TOR) pathway has been identified as a key requirement for the activation of egg development after a blood meal. We report the characterization of the TOR kinase and one of its major downstream targets, S6 kinase, of the yellow fever mosquito Aedes aegypti during egg development in adult females. Both TOR and S6K mRNA are expressed at high levels in the ovaries and in lower levels in fat body and other tissues. After a blood meal, the subcellular localization of TOR shifts from the cytoplasm to the plasma membrane of fat body cells. By detecting phosphothreonine 388 of mosquito S6 kinase, we show that TOR activity strongly increases in fat body and ovaries after a blood meal in vivo. Furthermore, phosphorylation of S6 kinase increases in in vitro cultured fat bodies after stimulation with amino acids. This increase is sensitive to the TOR inhibitor rapamycin in a concentration-dependent manner but not to the phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase-related kinase inhibitor LY294002, the MAPK inhibitor PD98059, or the translational inhibitor cycloheximide. RNA interference-mediated reduction of S6 kinase strongly inhibits the amino acid-induced up-regulation of the major yolk protein vitellogenin in vitro and effectively disrupts egg development after a blood meal in vivo. Our data show that TOR-dependent activation of S6 kinase is a central step in the transduction of nutritional information during egg development in mosquitoes.