Myeloid related protein induces muscle derived inflammatory mediators in juvenile dermatomyositis

Introduction

The aetiopathogenesis of juvenile dermatomyositis (JDM) remains poorly understood. In particular the contribution of monocytes or macrophages, which are frequently observed to be an infiltrate within muscle tissue very early in the disease process, is unknown. We hypothesised that these cells secrete the pro-inflammatory myeloid related protein (MRP) 8/14 which may then contribute to muscle pathology in JDM.

Methods

In this study of 56 JDM patients, serum MRP8/14 levels were compared with clinical measures of disease activity. Muscle biopsies taken early in disease were assessed by immunohistochemistry to determine the frequency and identity of MRP-expressing cells. The effects of MRP stimulation and endoplasmic reticulum (ER) stress on muscle were tested in vitro. Serum or supernatant levels of cytokines were analyzed by multiplex immunoassay.

Results

Serum MRP8/14 correlated with physician’s global assessment of disease activity in JDM (R = 0.65, p = 0.0003) and muscle strength/endurance, childhood myositis assessment score (CMAS, R = −0.55, p = 0.004). MRP8/14 was widely expressed by CD68+ macrophages in JDM muscle tissue. When cultured with human myoblasts, MRP8 led to the secretion of MCP-1 and IL-6, which was enhanced by ER stress. Both inflammatory mediators were detected in significantly higher levels in the serum of JDM patients compared to healthy controls.

Conclusions

This study is the first to identify serum MRP8/14 as a potential biomarker for disease activity in JDM. We propose that tissue infiltrating macrophages secreting MRP8/14 may contribute to myositis, by driving the local production of cytokines directly from muscle.     

Molecular diagnosis of apple virus and viroid pathogens from India

Apple is known to be susceptible to various virus and viroid pathogens. Symptomatic apple cultivars and rootstocks were collected and analyzed by ELISA and then through RT-PCR. The study reports the presence of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), the major apple viruses and Prunus necrotic ringspot virus (PNRSV), a minor apple virus, at the molecular level in India. Apple scar skin viroid (ASSVd) infection was also confirmed at the molecular level. Sporadic incidences of Tomato ringspot virus and Arabis mosaic virus infections were also detected by ELISA in nursery plants.     

Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of chloroquine in dog plasma

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3-->247.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0-489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0-489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine.     

Uterovaginal packing with rolled gauze in postpartum hemorrhage

Management options for postpartum hemorrhage (PPH) include oxytocics, prostaglandins, genital tract exploration, ligation or angiographic embolization of uterine/internal iliac arteries, and hysterectomy. After excluding uterine rupture, genital tract lacerations, and retained placental tissue, efforts are directed toward contracting the uterus by bimanual compression and oxytocics. If these are not successful, one must resort to surgical techniques. At this stage, an alternative option to remember is uterovaginal packing. Easy and quick to perform, it may be used to control bleeding by tamponade effect and stabilize the patient until a surgical procedure is arranged. Uterovaginal packing may sometimes obviate the need for surgery altogether. Two cases, a primary and a secondary PPH, managed recently with uterovaginal packing are reported. Despite concerns about concealed hemorrhage or the development of infection with this intervention, none of these problems were encountered, and uterine packing was successful even in the case of secondary PPH with documented infection.     

Weekly osimertinib dosing prevents EGFR mutant tumor cells destined to home mouse lungs

The recently conducted ADAURA trial concludes daily dosing of adjuvant osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), improves disease-free survival with stage IB/II/IIIA EGFR -mutated non-small cell lung cancer patients in comparison to placebo. We have developed a preclinical orthotopic mouse model, using luciferase tagged lung adenocarcinoma cells harboring EGFR TKI sensitive exon 19 deletion to model and extend trial implications comparing a weekly vs daily dosing outcome of osimertinib to a first-generation TKI- erlotinib. We find that 100% of mice in both the groups receiving osimertinib daily or weekly before injection of cells show a complete absence of homing of cells in mice''s lungs from day three until day 18 post-injection of cells. On the other hand, 25% and 75% of mice receiving erlotinib daily and weekly before injecting cells show homing of cells to the lungs. The tumors observed in the lungs, when dissected at day 30, confirmed the colonization of the injected cells homing to the organ. Thus, our study establishes the efficacy of pretreatment with osimertinib in reducing tumor cells'' homing to mouse lungs in an in vivo mouse model.     

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.     

Expression of recombinant Apple chlorotic leaf spot virus coat protein in heterologous system: production and use in immunodiagnosis

Expression condition for maximum recovery of recombinant Apple chlorotic leaf spot virus (ACLSV) coat protein was standardized. The in vitro expressed fusion protein with 6xHis tag (~43 Kd) was purified from inclusion bodies and used as an antigen for raising polyclonal antiserum in rabbit. This antiserum consistently detected ACLSV in pome and stone fruits as well as herbaceous host plants by direct double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and direct tissue blot immunoassay (DTBIA). The conditions for immuno-capture RT-PCR (IC-RT-PCR) were also standardized.     

Modeling and experimental analyses reveals signaling plasticity in a bi-modular assembly of CD40 receptor activated kinases

Depending on the strength of signal dose, CD40 receptor (CD40) controls ERK-1/2 and p38MAPK activation. At low signal dose, ERK-1/2 is maximally phosphorylated but p38MAPK is minimally phosphorylated; as the signal dose increases, ERK-1/2 phosphorylation is reduced whereas p38MAPK phosphorylation is reciprocally enhanced. The mechanism of reciprocal activation of these two MAPKs remains un-elucidated. Here, our computational model, coupled to experimental perturbations, shows that the observed reciprocity is a system-level behavior of an assembly of kinases arranged in two modules. Experimental perturbations with kinase inhibitors suggest that a minimum of two trans-modular negative feedback loops are required to reproduce the experimentally observed reciprocity. The bi-modular architecture of the signaling pathways endows the system with an inherent plasticity which is further expressed in the skewing of the CD40-induced productions of IL-10 and IL-12, the respective anti-inflammatory and pro-inflammatory cytokines. Targeting the plasticity of CD40 signaling significantly reduces Leishmania major infection in a susceptible mouse strain. Thus, for the first time, using CD40 signaling as a model, we show how a bi-modular assembly of kinases imposes reciprocity to a receptor signaling. The findings unravel that the signalling plasticity is inherent to a reciprocal system and that the principle can be used for designing a therapy.