Extracellular matrix-modulated differential invasion of human meningioma cell lines in vitro

The in vitro invasive behaviour of six meningioma cell lines of various histological sub-type and grade was assessed using Boyden chemotaxis chambers ('Transwell' units) precoated with various extracellular matrix proteins. The cell lines included a benign meningothelial (IPGS), two benign transitional (IPCBR and IPGC), one atypical (IPIH) and two malignant (IPSE and IPIR) meningiomas. IPGC was a recurrent tumour. The results showed that IPCBR was most invasive through laminin and vitronectin. IPIH was moderately invasive through collagen type IV, laminin, vitronectin and fibronectin. However, both IPSE and IPIR were less invasive than IPIH whereas, IPGS was least invasive of all. Moreover, laminin was the most permissive extracellular matrix protein for most cell lines and collagen type IV, the least permissive. These results show that there is a differential in vitro invasive behaviour of cell lines derived from different histological types of meningiomas according to extracellular matrix substrate and suggests that invasion and migration of meningiomas in situ might be modulated by various extracellular components.     

Calcinosis in juvenile dermatomyositis

A retrospective study of the radiographs and clinical records of 39 children with dermatomyositis, followed up at a referral centre for a mean period of eight years, revealed calcinosis in 29 children (18 giris, 11 boys). Clinical presentation of calcinosis was unpredictable and variable, ranging from four months to 12 years after onset of disease. Subcutaneous calcilication, often associated with subsequent ulceration of overlying skin, was observed more frequently than calcinosis in intermuscular fascial planes (29:7). Subcutaneous calcinosis was demonstrated most commonly about the knees and elbows and in the fingers and toes. Intermuscular calcinosis affected the fascial planes around the quadriceps, deltoid, biceps, and the gastrocnemius/soleus muscles.Calcinosis, predominantly of the subcutaneous lesions, regressed spontaneously in eight of 11 children where this could be assessed over a one to four year period. This high rate of spontaneous resolution must complicate evaluation of the efficacy of proposed anti-calcinotic treatments in juvenile dermatomyositis.While no apparent relationship was established between subcutaneous calcinosis, which was present in all 29 of the affected patients, and disease activity or severity, all seven children with the additional, extensive, and classical intermuscular fascial plane calcification developed this complication early and had severe disease.These findings are discussed in relation to previous studies.     

Lacrimal canaliculitis as a cause of recurrent conjunctivitis

BackgroundBacterial conjunctivitis is routinely managed by optometrists. This case report describes a case of lacrimal canaliculitis, which is a cause for nonresolving conjunctivitis and is commonly misdiagnosed.Case ReportA 48-year-old man presented with a 5-month history of chronic conjunctivitis in the right eye. This was diagnosed as bacterial in etiology but had a short-lived response to various topical antibiotics. Examination found signs of canaliculitis, and Actinomyces israelii was cultured from the discharge. This was treated successfully by canaliculotomy with curettage followed by a postoperative course of phenoxymethylpenicillin.ConclusionsIt is important to consider the diagnosis of lacrimal canaliculitis in any patient with chronic or recurrent conjunctivitis. Greater awareness of this condition would prevent misdiagnosis and delays in referral for definitive treatment.     

Evaluation of beta defensin 2 production by chicken heterophils using direct MALDI mass spectrometry

Beta defensins (BD) are cysteine rich, cationic antimicrobial peptides (AMP) produced mainly by epithelial and myeloid cells such as neutrophils. In birds, the neutrophil equivalent heterophils produce avian beta defensins (AvBD) of which AvBD2 is the major isoform. Heterophils recognize pathogens or their derived products through a series of pattern recognition receptors called toll-like receptors (TLR) leading to their antimicrobial activities. This work is the first report of TLR modulation of AvBD2 expression in chickens. To measure the effect of TLR activation on AvBD2 production, the heterophils were cultured with different TLR agonists for 6 h. Modulation of AvBD2 levels by TLR activation was measured using direct MALDI mass spectrometry without stable isotopic labeling or chromatographic separation. Chemical modification of the conditioned media was performed using reduction/alkylation with dithiothreitol/iodoacetamide to distinguish TLR treated AvBD2 (reduced/alkylated) from controls (non-reduced). Changes in corrected ion intensity ratios were assumed to reflect AvBD2 modulation in heterophils upon activation with different TLR agonists. In general, TLR agonists increased AvBD2 production with LPS showing the greatest induction and CpG-ODN showing little or no effect. These data show that the direct MALDI-MS coupled with reduction/alkylation may provide a rapid relative quantitative approach to the measurement of agonist-induced differential expression of AvBD2.     

Tofacitinib, a Janus Kinase Inhibitor Demonstrates Efficacy in an IL-15 Transgenic Mouse Model that Recapitulates Pathologic Manifestations of Celiac Disease

Celiac disease (CD) is an immune-mediated, inflammatory disorder of the small intestines with a defined genetic etiological component associated with the expression of HLA-DQ2 and/or HLA-DQ8 haplotypes. The dietary consumption of gluten-rich cereals triggers a gluten-specific immune response in genetically susceptible individuals leading to a spectrum of clinical manifestations ranging from an inapparent subclinical disease, to overt enteropathy that can in some individuals progress to enteropathy-associated T cell lymphoma (EATL). The tissue-destructive pathologic process of CD is driven by activated NK-like intraepithelial CD8⁺ lymphocytes and the proinflammatory cytokine IL-15 has emerged to be pivotal in orchestrating this perpetual tissue destruction and inflammation. Moreover, transgenic mice that over-express human IL-15 from an enterocyte-specific promoter (T3^b-hIL-15 Tg) recapitulate many of the disease-defining T and B cell-mediated pathologic features of CD, further supporting the evolving consensus that IL-15 represents a valuable target in devising therapeutic interventions against the form of the disease that is especially refractory to gluten-free diet. In the present study, we evaluated the potential efficacy of tofacitinib, a pan-JAK inhibitor that abrogates IL-15 signaling, as a therapeutic modality against CD using T3^b-hIL-15 Tg mice. We demonstrate that tofacitinib therapy leads to a lasting reversal of pathologic manifestations in the treated mice, thereby highlighting the potential value of tofacitininb as a therapeutic modality against refractory CD for which no effective therapy exists currently. Additionally, the visceral adiposity observed in the tofacitinib-treated mice underscores the importance of continued evaluation of the drug’s impact on the lipid metabolism.     

Physical and Biochemical Characterization of Chemically Treated Pollen Shells for Potential Use in Oral Delivery of Therapeutics

Allergen-free pollen shells obtained from natural pollen grains have recently attracted attention as microcapsules for oral therapeutic delivery. We have recently developed a chemical treatment method that enables successful retrieval of hollow pollen shells from diverse species. A comprehensive characterization is critical to characterize the effects of chemical treatment which will not only benchmark the pollen treatment process but can also lay the foundation of quality control procedures to check allergen-removal efficiency during pollen treatment. Therefore, in this study, we followed the effects of chemical treatment on 4 different pollen species using electron microscopy, elemental analysis, gel electrophoresis, confocal microscopy, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. These analyses revealed that acetone treatment removed lipids from the pollen surface. Phosphoric acid treatment removed proteins and nucleic acids from the pollen core and transformed esters into carboxylic acids. Potassium hydroxide hydrolysis changed carbohydrate composition of the pollen wall. Chemically treated pollen shells exhibited hydroxyl and carboxyl functional groups on their surface. Overall, we propose that confocal microscopy could be used as a rapid scanning technique to visualize the removal of biomolecules, whereas Fourier-transform infrared combined with gel electrophoresis could be used as a more objective approach for analysis and benchmarking.     

The flavoprotein FOXRED2 reductively activates nitro-chloromethylbenzindolines and other hypoxia-targeting prodrugs

The nitro-chloromethylbenzindoline prodrug SN29428 has been rationally designed to target tumour hypoxia. SN29428 is metabolised to a DNA minor groove alkylator via oxygen-sensitive reductive activation initiated by unknown one-electron reductases. The present study sought to identify reductases capable of activating SN29428 in tumours. Expression of candidate reductases in cell lines was modulated using forced expression and, for P450 (cytochrome) oxidoreductase (POR), by zinc finger nuclease-mediated gene knockout. Affymetrix microarray mRNA expression of flavoreductases was correlated with SN29428 activation in a panel of 23 cancer cell lines. Reductive activation and cytotoxicity of prodrugs were measured using mass spectrometry and antiproliferative assays, respectively. SN29428 activation under hypoxia was strongly attenuated by the pan-flavoprotein inhibitor diphenyliodonium, but less so by knockout of POR suggesting other flavoreductases contribute. Forced expression of 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), as well as POR, increased activation of SN29428 in hypoxic HCT 116 cells. SN29428 activation strongly correlated with expression of POR and also FAD-dependent oxidoreductase domain containing 2 (FOXRED2), in cancer cell lines. This association persisted after removing the effect of POR enzyme activity using first-order partial correlation. Forced expression of FOXRED2 increased SN29428 activation and cytotoxicity in hypoxic HEK293 cells and also increased activation of hypoxia-targeted prodrugs PR-104A, tirapazamine and SN30000, and increased cytotoxicity of the clinical-stage prodrug TH-302. Thus this study has identified three flavoreductases capable of enzymatically activating SN29428, one of which (FOXRED2) has not previously been implicated in xenobiotic metabolism. These results will inform future development of biomarkers predictive of SN29428 sensitivity.