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Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment.

Abstract Background Opioid abuse is widespread in North America and the over‐prescription of opioids are a contributing factor. The goal of this prospective study was to quantify over‐prescription rates, evaluate postoperative experiences of pain, and understand the impact of peri‐operative factors such as adequate pain counseling and use of non‐opioid analgesia. Methods Consecutive recruitment of patients undergoing head and neck endocrine surgery was undertaken from January 1st 2020 to December 31st 2021 at four Canadian hospitals in Ontario and Nova Scotia. Postoperative tracking of pain levels and analgesic requirements were employed. Chart review and preoperative and postoperative surveys provided information on counseling, use of local anesthesia, and disposal plans. Results A total of 125 adult patients were included in the final analysis. Total thyroidectomy was the most common procedure (40.8%). Median use of opioid tablets was 2 (IQR 0–4), with 79.5% of prescribed tablets unused. Patients who reported inadequate counseling (n = 35, 28.0%) were more likely to use opioids (57.2% vs. 37.8%, p

OBJECTIVES/GOALS: LSD1 is a histone demethylase important in GBM regulation. Our goal is to design a therapeutic strategy for LSD1 inhibitors to meet clinical needs in GBM. Despite the abundance of LSD1 inhibitors, resistance emerges in GBM mouse models. We aim to understand the relevance of proliferative signaling pathways, such as MAPK, in LSD1 inhibitor resistance. METHODS/STUDY POPULATION: Following LSD1 knockdown in GBM cells, we determined differentially expressed genes using RNA-seq and gene set enrichment analysis (GSEA). Kinase signaling processes enriched for LSD1 expression were identified. Utilizing western blot, we assessed LSD1’s impact on MAPK signaling in patient-derived GBM stem cells (GSCs) and pediatric high-grade glioma cell models. Pharmacological evaluation of LSD1 involved five inhibitor candidates. Additionally, we explored LSD1 inhibition in combination with brain penetrant kinase inhibitors, osimertinib and ulixertinib, directed against the epidermal growth factor receptor (EGFR) and MAPK, respectively. The treatment combinations were assessed at multiple concentrations and analyzed using SynergyFinder. RESULTS/ANTICIPATED RESULTS: Pharmacological LSD1 inhibition after 24 hours induced increased phosphorylated ERK1/2 across multiple glioma cell lines. Concurrent LSD1 and EGFR/MAPK inhibition demonstrated improvedin vitro efficacy compared to individual agents. Notably, the combination of Iadademstat (ORY-1001) and osimertinib demonstrated the highest synergy score of 37.2 using the bliss synergy model in the GSC17s. Furthermore, 11 out of the 12 combination treatments tested had a synergistic relationship, with bliss synergy scores greater than 10. DISCUSSION/SIGNIFICANCE: Our study addresses the pressing need for novel therapeutic strategies in GBM. We leveraged pharmacological tools of LSD1 inhibition to determine how they could be used most effectively, revealing kinase inhibition as a promising strategy with demonstrated in vitro efficacy. Future efforts will focus on validating these findingsin vivo.

Methotrexate is a commonly used agent in the treatment of many malignancies and rheumatologic/inflammatory diseases. Working by inhibiting dihydrofolate reductase and thereby preventing eventual formation of tetrahydrofolate, methotrexate inhibits synthesis of purines and thymidylate, therefore disabling a malignant cell’s ability to replicate. While it is able to effectively do this, methotrexate also holds potential for significant toxicity. Therefore, serum methotrexate monitoring is of utmost importance when administering the drug, particularly when high doses are used. Although there are several different measurement systems, the immunoassay is a commonly used monitoring system that may be prone to interference when using agents with similar carbon backbone as methotrexate, including folinic acid (leucovorin) at high doses, as well as in the setting of glucarpidase use and consequent methotrexate breakdown. However, adjusting leucovorin dosing policy and being aware of the potential of the immunoassay to be “confused” by similar molecules have allowed for the efficient and effective use of the immunoassay while preventing prolonged hospital stays at our institution.

Abstract Background Intra-operative internal carotid artery (ICA) injury during transnasal endoscopic surgery is a potentially catastrophic event. Such an injury is life-threatening in the immediate setting, with a reported peri-operative mortality rate of 10%. Nasal packing, muscle patches, direct vessel closure, and endovascular techniques have been described as useful strategies for managing ICA bleeds. The objective of this study was to develop a formalized management protocol for intra-operative ICA injury through engagement with a multi-disciplinary panel. Methods A modified Delphi method including literature review, iterative rounds of stakeholder feedback, and expert panel discussions was used to develop a management protocol for ICA injury during transnasal endoscopic surgery. The 10-person multi-disciplinary panel included otolaryngologists, neurosurgeons, interventional neuroradiologists, anesthesiologists, and operating room nursing staff. Results After three rounds of stakeholder engagement with the expert panel, consensus was reached on important elements to include within the protocol. The protocol was divided in three categories: Alert, Control, and Transfer. ‘Alert’ focusses on early communication with anesthesia and nursing staff. ‘Control’ focusses on techniques to expose the injury and obtain hemostasis or adequate tamponade. Lastly, ‘Transfer’ describes the process of contacting neuro-interventional radiology and safely transferring the patient. A one-page handout of the protocol was developed for placement in operating theatres. Conclusion Due to the life-threatening nature of ICA injury, it is imperative that endoscopic sinus and skull base surgeons are prepared to manage this complication. Using a modified Delphi method with a multidisciplinary expert panel, a protocol for management of intra-operative ICA injury was developed. Graphical Abstract

Abstract Background Myxofibrosarcoma is a common soft tissue sarcoma of the extremities, which occurs very rarely in the thyroid gland. Case presentation We report the case of a 61-year-old male who presented with a swelling of the left side of the neck and a newly emerged hoarseness. Ultrasound depicted a hypoechoic thyroid nodule with microcalcifications that was highly suspicious for malignancy. He underwent a left hemithyroidectomy. Histopathological examination and immunohistochemical studies revealed a myxofibrosarcoma of the thyroid gland. Conclusion Myxofibrosarcoma of the thyroid gland is extremely rare. The diagnosis is based on histopathological features. Radical surgery achieving tumor-free resection margins remains the only chance for cure. However, the role of radiotherapy and/or chemotherapy is still under debate. Due to their high tendency for locoregional recurrence, a close follow-up after surgery is mandatory.

Abstract Background The rise in the use of prescription opioids for postoperative analgesia within surgery has mirrored an increased trend of opioid‐related morbidity within Canada and the United States. This study prospectively studied daily pain levels and medication requirements postoperatively in patients undergoing elective Otolaryngology—Head and Neck surgery procedures. Methods Patients were asked to prospectively document their pain level and medication use daily for 7 days postoperatively. A final survey was used to quantify unused medication left at home and clarify each patient's disposal plan. We included patients undergoing elective outpatient or short stay surgeries from three tertiary care centers in Toronto, Ontario from September 2016 to September 2017. Previous opioids users or patients suffering from chronic pain were excluded. Results A final cohort of 56 eligible adult patients were included in the study. The most common procedures were thyroidectomy (n = 19), endoscopic sinus surgery (n = 10), tympanoplasty/ossiculoplasty (n = 7), and cochlear implant (n = 5). Most patients received a prescription for acetaminophen/codeine (n = 29, 51.8%) or acetaminophen/oxycodone (n = 22, 39.3%) and used on average 29% of their initial prescription. Patients most commonly opted to keep their unused narcotics at home (n = 23, 41%). A total of 710 tablets of narcotics were overprescribed in our study population, 351 of which were kept in patients' home for future use. Conclusion There is a clear tendency to overestimate postoperative pain resulting in significant overprescription of opioids among Otolaryngologists.

ABSTRACT Biofilm and hypha formation are central to virulence of the fungal pathogen Candida albicans. The G1 cyclin gene HGC1 is required for hypha formation under diverse in vitro and in vivo growth conditions. Hgc1 is required for disseminated infection and is a linchpin in the argument that hyphal morphogenesis itself is required for pathogenicity. We report here that HGC1 is dispensable for hypha formation during biofilm formation both in vitro, under strong inducing conditions, and in vivo, in a mouse oropharyngeal candidiasis model. These findings are validated with two or more C. albicans isolates. Systematic screening of overexpressed cyclin genes indicates that CCN1 and CLN3 can compensate partially for Hgc1 function during biofilm growth. This conclusion is also supported by the severity of the hgc1Δ/Δ ccn1Δ/Δ double mutant biofilm defect. Our results suggest that hypha formation in biofilm is accomplished by combined action of multiple cyclins, not solely by Hgc1. IMPORTANCE The HGC1 gene encodes a cyclin that is required for virulence of the fungal pathogen Candida albicans. It is required to produce the elongated hyphal filaments of free-living planktonic cells that are associated with virulence. Here, we show that HGC1 is not required to produce hyphae in the alternative growth form of a biofilm community. We observe Hgc1-independent hyphae in two infection-relevant situations, biofilm growth in vitro and biofilm-like oropharyngeal infection. Our analysis suggests that hypha formation in the biofilm state reflects combined action of multiple cyclins.

Abstract Limited information is available on the efficacy of front‐line bendamustine and rituximab (BR) in chronic lymphocytic leukemia (CLL) with reduced renal function or coexisting conditions. We therefore analyzed a cohort of real‐world patients and performed a matched adjusted indirect comparison with a cohort of patients treated with ibrutinib. One hundred and fifty‐seven patients with creatinine clearance (CrCl) 6 were treated with BR. The median age was 72 years; 69% of patients had ≥2 comorbidities and the median CrCl was 59.8 mL/min. 17.6% of patients carried TP53 disruption. The median progression‐free survival (PFS) was 45 months; TP53 disruption was associated with a shorter PFS (P = 0.05). The overall survival (OS) at 12, 24, and 36 months was 96.2%, 90.1%, and 79.5%, respectively. TP53 disruption was associated with an increased risk of death (P = 0.01). Data on 162 patients ≥65 years treated with ibrutinib were analyzed and compared with 165 patients ≥65 years treated with BR. Factors predicting for a longer PFS at multivariable analysis in the total patient population treated with BR and ibrutinib were age (HR 1.06, 95% CI 1.02‐1.10, P

The Chronographia of Michael Psellos (1018–1081) reveals a limited interest in nations and minorities within and without the Byzantine Empire. He had access to information about these peoples either indirectly (1018–1042) or more directly (1042–1078). He has a greater understanding of their complexity, especially between 1042–1059 when his friend Constantine Leichoudes was mesazon. Psellos refers to nations and minorities in his Chronographia through the prism of the imperial court at Constantinople.

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP–NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin’s lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN–PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

We present the case of a 55-year-old woman who presented with laboratory studies concerning for acute myeloid leukemia (AML) as well as obstructive cholestasis. In similar previously reported cases, concerns of chemotherapy toxicity exacerbated by liver dysfunction or concerns of untreated, concurrent cholecystitis in a neutropenic patient often delay initiation of chemotherapy for full medical workup. At admission, our patient was started on the cytoreductive agent hydroxyurea. By day 10 of her medical workup, her liver function had improved with total bilirubin levels normalizing. At that time, full-dose 7 + 3 induction with cytarabine and daunorubicin was then initiated.

Plasmacytoid dendritic cell neoplasms are aggressive and rare hematologic malignancies characterized by clonal expansion of plasmacytoid dendritic cells with frequent cutaneous involvement. The pathogenesis is not well established, and it shows enhanced expression of CD56, CD4 and CD123 detected by flow cytometry and immunohistochemistry. We report a case report of this rare disease in a hispanic child with complete remission after using a protocol for high-risk acute lymphoblastic leukemia.

ATRX inactivation frequently occurs in glioma. Here, the authors explore the role of ATRX inactivation in oncogenesis, highlighting ATRX deficiency driven epigenomic changes that influence the expression of genes crucial to the oncogenic phenotype.

Objective: Collisions are more common among drivers with type 1 diabetes than among their nondiabetic spouses. This increased risk appears to be attributable to a subgroup of drivers with type 1 diabetes. The hypothesis tested is that this vulnerable subgroup is more at risk for hypoglycemia and its disruptive effects on driving.Research Design and Methods: Thirty-eight drivers with type 1 diabetes, 16 with (+history) and 22 without (-history) a recent history of recurrent hypoglycemia-related driving mishaps, drove a virtual reality driving simulator and watched a videotape of someone driving a simulator for 30-min periods. Driving and video testing occurred in a double-blind, randomized, crossover manner during euglycemia (5.5 mmol/l) and progressive hypoglycemia (3.9-2.5 mmol/l). Examiners were blind to which subjects were +/-history, whereas subjects were blind to their blood glucose levels and targets.Results: During euglycemia, +history participants reported more autonomic and neuroglycopenic symptoms (P≤0.01) and tended to require more dextrose infusion to maintain euglycemia with the same insulin infusion (P<0.09). During progressive hypoglycemia, these subjects demonstrated less epinephrine release (P=0.02) and greater driving impairments (P=0.03).Conclusions: Findings support the speculation that there is a subgroup of type 1 diabetic drivers more vulnerable to experiencing hypoglycemia-related driving mishaps. This increased vulnerability may be due to more symptom "noise" (more symptoms during euglycemia), making it harder to detect hypoglycemia while driving; possibly greater carbohydrate utilization, rendering them more vulnerable to experiencing hypoglycemia; less hormonal counterregulation, leading to more profound hypoglycemia; and more neuroglycopenia, rendering them more vulnerable to impaired driving. [ABSTRACT FROM AUTHOR]

The Synodikon of Orthodoxy, in its earliest version (Synodikon of Alexius Studites), has at least five different layers of composition. Beside the original one of 843/844, there is one after 845, another after 870, a fourth after 925 and finally one between 1034–1043. Since each date represents a post quem, they could be grouped together. However, the layers of composition represent different interests and objectives and therefore are difficult to conflate. On the contrary, each stratum reveals that by the 11th century the characteristic of the Synodikon was to unify different objectives and strategies into one text. It is also for this reason that the text continued to expand after Alexius Studites’ version of 1034–1043.

The purpose of this study was to determine objectively the relationships between changes in the usual amount of insulin injected, food eaten, and exercise performed, and the subsequent occurrence of low blood glucose (< 3.9 mM) in adults with IDDM and varying degrees of hypoglycemic awareness and metabolic control. Subjects used a handheld computer to record whether their most recent insulin, food, and exercise had been omitted or were greater than, less than, or about the same as usual following every measured blood glucose level of < 3.9 mM and > 5.6 mM. Responses for each self-management behavior were compared for the two blood glucose ranges. Food was omitted more frequently prior to a low glucose reading and exercise was omitted more frequently prior to a high glucose reading. More insulin, less food, and more exercise each were associated with low glucose levels. These findings underscore the importance of traditional diabetes education. [ABSTRACT FROM AUTHOR]

Venetoclax is a BCL2 inhibitor approved for 17p-deleted relapsed/refractory chronic lymphocytic leukemia with activity following kinase inhibitors. We conducted a multicenter retrospective cohort analysis of patients with chronic lymphocytic leukemia treated with venetoclax to describe outcomes, toxicities, and treatment selection following venetoclax discontinuation. A total of 141 chronic lymphocytic leukemia patients were included (98% relapsed/refractory). Median age at venetoclax initiation was 67 years (range 37-91), median prior therapies was 3 (0-11), 81% unmutated IGHV, 45% del(17p), and 26.8% complex karyotype (≥ 3 abnormalities). Prior to venetoclax initiation, 89% received a B-cell receptor antagonist. For tumor lysis syndrome prophylaxis, 93% received allopurinol, 92% normal saline, and 45% rasburicase. Dose escalation to the maximum recommended dose of 400 mg daily was achieved in 85% of patients. Adverse events of interest included neutropenia in 47.4%, thrombocytopenia in 36%, tumor lysis syndrome in 13.4%, neutropenic fever in 11.6%, and diarrhea in 7.3%. The overall response rate to venetoclax was 72% (19.4% complete remission). With a median follow up of 7 months, median progression free survival and overall survival for the entire cohort have not been reached. To date, 41 venetoclax treated patients have discontinued therapy and 24 have received a subsequent therapy, most commonly ibrutinib. In the largest clinical experience of venetoclax-treated chronic lymphocytic leukemia patients, the majority successfully completed and maintained a maximum recommended dose. Response rates and duration of response appear comparable to clinical trial data. Venetoclax was active in patients with mutations known to confer ibrutinib resistance. Optimal sequencing of newer chronic lymphocytic leukemia therapies requires further study.

The Candida albicans RHR2 gene, which specifies a glycerol biosynthetic enzyme, is required for biofilm formation in vitro and in vivo. Prior studies indicate that RHR2 is ultimately required for expression of adhesin genes, such as ALS1. In fact, RHR2 is unnecessary for biofilm formation when ALS1 is overexpressed from an RHR2-independent promoter. Here, we describe two additional biological processes that depend upon RHR2: invasion into an abiotic substrate and pathogenicity in an abdominal infection model. We report here that abiotic substrate invasion occurs concomitantly with biofilm formation, and a screen of transcription factor mutants indicates that biofilm and hyphal formation ability correlates with invasion ability. However, analysis presented here of the rhr2Δ/Δ mutant separates biofilm formation and invasion. We found that an rhr2Δ/Δ mutant forms a biofilm upon overexpression of the adhesin gene ALS1 or the transcription factor genes BRG1 or UME6. However, the biofilm-forming strains do not invade the substrate. These results indicate that RHR2 has an adhesin-independent role in substrate invasion, and mathematical modeling argues that RHR2 is required to generate turgor. Previous studies have shown that abdominal infection by C. albicans has two aspects: infection of abdominal organs and persistence in abscesses. We report here that an rhr2Δ/Δ mutant is defective in both of these infection phenotypes. We find here that overexpression of ALS1 in the mutant restores infection of organs, but does not improve persistence in abscesses. Therefore, RHR2 has an adhesin-independent role in abdominal infection, just as it does in substrate invasion. This report suggests that RHR2, through glycerol synthesis, coordinates adherence with host- or substrate-interaction activities that enable proliferation of the C. albicans population.

Biofilm formation on implanted medical devices is a major source of lethal invasive infection by Candida albicans. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A cak1 diminished expression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the transcription factors Bcr1, Brg1, Efg1, Tec1, and Ume6. In the cak1 DX background, the dependence of filamentation-associated gene expression on each transcription factor is substantially relieved. The unexpected bypass of filamentation-associated gene expression activators has the functional consequence of enabling biofilm formation in the absence of Bcr1, Brg1, Tec1, Ume6, or in the absence of both Brg1 and Ume6. It also enables filamentous cell morphogenesis, though not biofilm formation, in the absence of Efg1. Because these transcription factors are known to have shared target genes, we suggest that cell cycle/cell growth limitation leads to activation of several transcription factors, thus relieving dependence on any one.

Web-mapping has been widely used to facilitate citizen participation in smart cities. Web-mapping has evolved from 2D static maps towards more dynamic and immersive 3D worlds such as virtual globes and scenes. Although current technologies allow us to build multidimensional representations, there is still a lack of research studies on how to further leverage them to foster citizen participation. Information on space−time changes can be an important asset for a successful citizen participation process. Citizens may need to track the evolution of their city over space and time, and how their participation will impact the urban planning and decision-making process. Consequently, the main objective of this research study is to design and develop a multidimensional (2D, 3D, 4D) web-mapping platform where citizens can better assess and understand the spatiotemporal evolution of their cities. User testing was conducted to assess the multidimensional representation of the animations used, and the spatiotemporal mechanism and interface features. Results showed that it is recommended to integrate spatiotemporal simulations to citizen participation platforms so citizens can better assess the impacts of their choices. We also assessed that 3D does not always communicate information better than 2D. Future work will aim at testing the platform in a consultation process with a representative sample of participants.

Maximus drew on the writings of the pagan Proclus in order to articulate a passage of Dionysius the Areopagite so as to support a view of two ‘powers/energies’ of Christ.

Frederick Lansigan1, Diane M Stearns1, Francine Foss21Hematology/Oncology, Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA; 2Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USAAbstract: Denileukin diftitox (Ontak®) is indicated for the treatment of patients with persistent or recurrent cutaneous T-cell lymphoma (CTCL), a rare lymphoproliferative disorder of the skin. Denileukin diftitox was the first fusion protein toxin approved for the treatment of a human disease. This fusion protein toxin combines the IL2 protein with diphtheria toxin, and targets the CD25 subunit of the IL2 receptor, resulting in the unique delivery of a cytocidal agent to CD-25 bearing T-cells. Historically, immunotherapy targeting malignant T-cells including monoclonal antibodies has been largely ineffective as cytocidal agents compared to immunotherapy directed against B-cells such as rituximab. This review will summarize the development of denileukin diftitox, its proposed mechanism of action, the pivotal clinical trials that led to its FDA approval, the improvements in quality of life, and the common toxicities experienced during the treatment of patients with CTCL. CTCL is often a chronic progressive lymphoma requiring the sequential use of treatments such as retinoids, traditional chemotherapy, or biological response modifiers. The incorporation of the immunotoxin denileukin diftitox into the sequential or combinatorial treatment of CTCL will also be addressed.Keywords: denileukin diftitox, cutaneous T-cell lymphoma, fusion protein toxin

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcγRIIA or both FcγRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.