Human dendritic cells express the complement receptor immunoglobulin which regulates T cell responses

The B7 family-related protein V-set and Ig containing 4 (VSIG4), also known as Z39Ig and Complement Immunoglobulin Receptor (CRIg), is the most recent of the complement receptors to be identified, with substantially distinct properties from the classical complement receptors. The receptor displays both phagocytosis–promoting and anti-inflammatory properties. The receptor has been reported to be exclusively expressed in macrophages. We now present evidence, that CRIg is also expressed in human monocyte-derived dendritic cells (MDDC), including on the cell surface, implicating its role in adaptive immunity. Three CRIg transcripts were detected and by Western blotting analysis both the known Long (L) and Short (S) forms were prominent but we also identified another form running between these two. Cytokines regulated the expression of CRIg on dendritic cells, leading to its up- or down regulation. Furthermore, the steroid dexamethasone markedly upregulated CRIg expression, and in co-culture experiments, the dexamethasone conditioned dendritic cells caused significant inhibition of the phytohemagglutinin-induced and alloantigen-induced T cell proliferation responses. In the alloantigen-induced response the production of IFNγ, TNF-α, IL-13, IL-4, and TGF-β1, were also significantly reduced in cultures with dexamethasone-treated DCs. Under these conditions dexamethasone conditioned DCs did not increase the percentage of regulatory T cells (Treg). Interestingly, this suppression could be overcome by the addition of an anti-CRIg monoclonal antibody to the cultures. Thus, CRIg expression may be a control point in dendritic cell function through which drugs and inflammatory mediators may exert their tolerogenic- or immunogenic-promoting effects on dendritic cells.Usma Munawara, Khalida Perveen, Annabelle G. Small, Trishni Putty, Alex Quach, Nick N. Gorgani, Charles S. Hii, Catherine A. Abbott and Antonio Ferrant

Phosphoproteomics Identifies Oncogenic Ras Signaling Targets and Their Involvement in Lung Adenocarcinomas

Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer

Delayed blood processing leads to rapid deterioration in the measurement of the neutrophil respiratory burst by the dihydrorhodamine-123 reduction assay

BACKGROUND:Neutrophils ex vivo in whole blood specimens are widely understood to decay rapidly when compared to other leukocytes, requiring assessment of neutrophil activity to be performed shortly after blood collection. There is a disparity in evidence for decay rates in measurements and recommended time-frames for assaying neutrophil parameters in particular assays following blood collection. We, therefore, evaluated the decline in the neutrophil respiratory burst, typically screened for assessing congenital NADPH oxidase defects, over a shorter time-course than previously published experiments. METHODS:The neutrophil respiratory burst was assessed by flow cytometric detection of DHR-123 oxidation to rhodamine-123 (Rho123), following stimulation of neutrophils by phorbol myristate acetate (PMA), in heparinized healthy donor blood specimens immediately following venipuncture, and then at 3 and 5 h later with ambient temperature or refrigerated specimen storage. RESULTS:A consistent time-dependent decline in the Rho123 fluorescence of PMA-stimulated neutrophils was detected in the healthy donor specimens, indicating a decay in respiratory burst activity. Neutrophil oxidative indexes calculated for half of the specimens at 3 and 5 h of age, fell below our normal laboratory lower limit. We also found that Rho123 histograms of PMA-stimulated neutrophils from stored healthy donor specimens have a risk of misinterpretation due to mimicking the appearance of histograms from carriers of CGD and other NADPH oxidase defects. Refrigeration of specimens did not significantly minimize decay. CONCLUSIONS:DHR assay of the neutrophil respiratory burst from blood specimens at 3 h post-venipuncture and beyond can generate unreliable clinical measurements due to decay. © 2019 International Clinical Cytometry Society.Alex Quach, Shannon Glowik, Trishni Putty, Antonio Ferrant

Process Integration for active polysilicon resonant microstructures

Microsensors based on active polysilicon resonant microstructures are attractive because of their wide dynamic range, high sensitivity and frequency shift output. In this paper, we discuss processing issues for integrating electrostatically-driven and -sensed polysilicon microstructures with on-chip nMOS device. Surface-micro-machining using sacrificial spacer layers is used to obtain relased microstructures. A novel feature is the use of rapid thermal annealing (RTA) for strain relief of the ion-implanted, phosphorous-doped polysilicon. Resonance frequencies of cantilever beams indicate a lower-bound Young's modulus of about 90 GPa and an upper-bound compressive residual strain of only 0.002%, indicating that RTA is potentially useful for strain relief.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27685/1/0000069.pd

Isolated anti-Ro52 identifies a severe subset of Sjögren's syndrome patients

INTRODUCTION: Serum autoantibodies targeting the SSA/Ro proteins are a key component of the classification criteria for the diagnosis of Sjögren's syndrome (SS). Most patients' serum reacts with both Ro60 and Ro52 proteins. Here we compare the molecular and clinical characteristics of patients diagnosed with SS with anti-Ro52 in the presence or absence of anti-Ro60/La autoantibodies. METHODS: A cross-sectional study was performed. Patients in the SS biobank at Westmead Hospital (Sydney, Australia) that were positive for anti-Ro52 were included and stratified based on the absence (isolated) or presence (combined) of anti-Ro60/La, measured by line immunoassay. We examined clinical associations and the serological and molecular characteristics of anti-Ro52 using ELISA and mass spectrometry in serological groups. RESULTS: A total of 123 SS patients were included for study. SS patients with isolated anti-Ro52 (12%) identified a severe serological subset characterised by higher disease activity, vasculitis, pulmonary involvement, rheumatoid factor (RhF) and cryoglobulinaemia. Serum antibodies reacting with Ro52 in the isolated anti-Ro52 subset displayed less isotype switching, less immunoglobulin variable region subfamily usage and a lower degree of somatic hypermutation than the combined anti-Ro52 subset. CONCLUSIONS: In our cohort of SS patients, isolated anti-Ro52 represents a severe subset of SS, and is associated with the presence of cryoglobulinaemia. We therefore provide clinical relevance to the stratification of SS patients by their sero-reactivities. It is possible that the autoantibody patterns may be immunological epiphenomena of the underlying disease process, and further work is required to unearth the mechanisms of the differential clinical phenotypes

Vitamin D upregulates the macrophage complement receptor immunoglobulin in innate immunity to microbial pathogens

Vitamin D deficiency remains a global concern. This 'sunshine' vitamin is converted through a multistep process to active 1,25-dihydroxyvitamin D₃ (1,25D), the final step of which can occur in macrophages. Here we demonstrate a role for vitamin D in innate immunity. The expression of the complement receptor immunoglobulin (CRIg), which plays an important role in innate immunity, is upregulated by 1,25D in human macrophages. Monocytes cultured in 1,25D differentiated into macrophages displaying increased CRIg mRNA, protein and cell surface expression but not in classical complement receptors, CR3 and CR4. This was associated with increases in phagocytosis of complement opsonised Staphylococcus aureus and Candida albicans. Treating macrophages with 1,25D for 24 h also increases CRIg expression. While treating macrophages with 25-hydroxyvitamin D3 does not increase CRIg expression, added together with the toll like receptor 2 agonist, triacylated lipopeptide, Pam3CSK4, which promotes the conversion of 25-hydroxyvitamin D₃ to 1,25D, leads to an increase in CRIg expression and increases in CYP27B1 mRNA. These findings suggest that macrophages harbour a vitamin D-primed innate defence mechanism, involving CRIg.Annabelle G. Small, Sarah Harvey, Jaspreet Kaur, Trishni Putty, Alex Quach, Usma Munawara, Khalida Perveen, Andrew McPhee, Charles S. Hii, Antonio Ferrant

Terapi Topikal Kombinasi Krim AsamTraneksamat 3%, Nikotinamid 3% dan Microneedling pada Pasien Melasma (Pilot Study)

Latar belakang : Melasma adalah hiperpigmentasi wajah pada wanita di area yang sering terpapar sinar matahari, bersifat kronis kambuhan. Saat ini formula Kligman masih menjadi pilihan terapi efektif tetapi mempunyai efek samping terutama pada pemakaian jangka lama. Kombinasi terapi asam traneksamat, nikotinamid dan microneedling merupakan terapi alternatif yang aman dan efektif untuk jangka lama. Tujuan : Untuk mengetahui keberhasilan terapi kombinasi topikal krim asam traneksamat 3%, nikotinamid 3% dan microneedling dalam menurunkan skor MASI dan indeks melanin pasien melasma. Kasus : Dua pasien perempuan umur 45 dan 44 tahun dengan keluhan utama bercak hitam di wajah. Pasien 1 dengan melasma tipe campuran dan pasien 2 dengan melasma tipe epidermal. Pasien diterapi topikal menggunakan krim kombinasi asam traneksamat 3% dan nikotinamid 3% dengan microneedling. Microneedling dilakukan pada minggu ke-0, ke-4 dan ke-8. Evaluasi terapi saat minggu ke-0, 4, 8 dan 12, dengan skor MASI dan indeks melanin menggunakan Mexameter®. Hasil : Terdapat penurunan nilai skor MASI dan indeks melanin. Simpulan : Terapi kombinasi krim asam traneksamat 3%, nikotinamid 3% dan microneedling dapat menurunkan skor MASI dan indeks melanin.Melasma is chronic and recurrent facial hyperpigmentation common in women and in areas frequently exposed to sunlight. Kligman formula therapy is still the effective treatment but has several side effects especially in long-term use. Combination of tranexamic acid, nicotinamide and microneedling is a safe and effective alternative for the long term therapy.Objective: To measure the effectiveness of topical combination therapy of 3% tranexamic acid cream, nicotinamide 3% and microneedling in reducing MASI score and melanin index in melasma patients. Cases: Two females aged 45 and 44 years with facial black spots. First patient diagnosed as mixed type melasma and 2nd patient with epidermal type melasma. Patients were treated with topical combination of 3% tranexamic acid and 3% nicotinamide cream with microneedling. Microneedling were done in the 0th, 4th and 8th weeks. Evaluation and measurement was done at weeks 0, 4, 8 and 12, with MASI scores and melanin index using Mexameter®. Results: There was a decrease in MASI score and melanin index.Conclusion: Combination therapy of tranexamic acid cream 3%, nicotinamide 3% and microneedling could reduce MASI score and melanin index