The First WHO International Standard for Harmonizing the Biological Activity of Bevacizumab.

Several Bevacizumab products are approved for clinical use, with many others in late-stage clinical development worldwide. To aid the harmonization of potency assessment across different Bevacizumab products, the first World Health Organization (WHO) International Standard (IS) for Bevacizumab has been developed. Two preparations of a Bevacizumab candidate and comparator were assessed for their ability to neutralize and bind vascular endothelial growth factor (VEGF) using different bioassays and binding assays in an international collaborative study. Relative potency estimates were similar across different assays for the comparator or the duplicate-coded candidate sample. Variability in relative potency estimates was reduced when the candidate standard was used for calculation compared with various in-house reference standards, enabling harmonization in bioactivity evaluations. The results demonstrated that the candidate standard is suitable to serve as an IS for Bevacizumab, with assigned unitages for VEGF neutralization and VEGF binding activity. This standard coded 18/210 was established by the WHO Expert Committee on Biological Standardization, which is intended to support the calibration of secondary standards for product development and lifecycle management. The availability of IS 18/210 will help facilitate the global harmonization of potency evaluation to ensure patient access to Bevacizumab products with consistent safety, quality and efficacy.

Quantification of Pancreatic Islets: Using Image Analysis Tools.

Histological image analysis is becoming an increasingly important tool for research in biological science. They are important in analyzing biological systems on various scales, from structural details to determination of number of cells, its area, localization, and concentration. This chapter focuses on analysis of pancreatic sections stained for insulin and glucagon using a commercially available software.

IL-33 stimulates expression of the GPR84 (EX33) fatty acid receptor gene and of cytokine and chemokine genes in human adipocytes.

Expression of GPCR fatty acid sensor/receptor genes in adipocytes is modulated by inflammatory mediators, particularly IL-1ß. In this study we examined whether the IL-1 gene superfamily member, IL-33, also regulates expression of the fatty acid receptor genes in adipocytes. Human fat cells, differentiated from preadipocytes, were incubated with IL-33 at three different dose levels for 3 or 24 h and mRNA measured by qPCR. Treatment with IL-33 induced a dose-dependent increase in GPR84 mRNA at 3 h, the level with the highest dose being 13.7-fold greater than in controls. Stimulation of GPR84 expression was transitory; the mRNA level was not elevated at 24 h. In contrast to GPR84, IL-33 had no effect on GPR120 expression. IL-33 markedly stimulated expression of the IL1B, CCL2, IL6, CXCL2 and CSF3 genes, but there was no effect on ADIPOQ expression. The largest effect was on CSF3, the mRNA level of which increased 183-fold over controls at 3 h with the highest dose of IL-33; there was a parallel increase in the secretion of G-CSF protein into the medium. It is concluded that in human adipocytes IL-33, which is synthesised in adipose tissue, has a strong stimulatory effect on the expression of cytokine and chemokine genes, particularly CSF3, and on the expression of GPR84, a pro-inflammatory fatty acid receptor.

Genetic homogeneity of adult Langerhans cell histiocytosis lesions: Insights from BRAFV600E mutations in adult populations.

Langerhans cell histiocytosis (LCH) is a heterologous disease with a recognized disparity in incidence, affected sites and prognosis between adults and children. The recent identification of BRAFV600E mutations in LCH prompted the investigation of the frequency of these mutations in adult and childhood disease with the involvement of single or multiple sites in the present study. The study analysed the BRAFV600E status in a cohort of adult LCH patients by DNA sequencing, and performed a broader meta-analysis of BRAFV600E mutations in LCH in order to investigate any association with disease site and severity. A review of the literature revealed that ~47% of lesions from cases of adult disease (patient age, >18 years) were V600E-positive compared with 53% in those under 18 years. When single and multiple site disease was compared, there was a slight increase in the former (61 vs. 51%, respectively). A greater difference was observed when high- and low-risk organs were compared; for example, 75% of liver biopsies (a high-risk organ) were reported to bear the mutation compared with 47% of lung biopsies. In the adult LCH population, DNA sequencing identified mutations in 38% of 29 individuals, which is slightly lower than the figure identified from the meta-analysis (in which a total of 132 individuals were sampled), although we this value could not be broken down by clinical status. Thus, V600E status at presentation in itself is not predictive of tumour course, but a considerable proportion of LCH patients may respond to targeted V600E therapies.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Collagen remodeling and peripheral immune cell recruitment characterizes the cutaneous Langerhans cell histiocytosis microenvironment.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare and potentially fatal disorder of unknown etiology arising from the accumulation of epidermal Langerhans-like cells in bone, skin, or other tissues. Tissue damage and morbidity results from lesional cytokine release, and we sought to investigate the LCH microenvironment using a combination of histological stains and immunohistochemistry. METHODS: CD1a immunoreactivity was used to identify lesional cells in archival paraffin-embedded samples of cutaneous LCH. A combined Orcein and Giemsa stain identified immune cells in general (particularly granulocytes and mast cells) and extracellular matrix (particularly elastic fibers), while CD3 and CD68 staining identified T cells and macrophages, respectively. Collagen synthesis was investigated with Herovici staining, which discriminates newly synthesized from mature collagen, while cross-polar microscopy of picrosirius-stained sections identified changes in matrix organization. RESULTS: Immune cells were consistently identified at the periphery of cutaneous LCH lesions. We quantified an increased number of thickened and disorganized elastic fibers surrounding lesions and an absence of elastic fibers within lesions. Furthermore, lesions exhibited a decrease in mature collagen fibers and a loss of supporting collagen matrix within lesions and compromised collagen integrity in adjacent tissue. CONCLUSIONS: Cutaneous LCH lesions are associated with the peripheral recruitment of a variety of immune cells and are consistently characterized by localized elastosis, collagen damage, and remodeling.

A novel method to assess collagen architecture in skin.

BACKGROUND: Texture within biological specimens may reveal critical insights, while being very difficult to quantify. This is a particular problem in histological analysis. For example, cross-polar images of picrosirius stained skin reveal exquisite structure, allowing changes in the basketweave conformation of healthy collagen to be assessed. Existing techniques measure gross pathological changes, such as fibrosis, but are not sufficiently sensitive to detect more subtle and progressive pathological changes in the dermis, such as those seen in ageing. Moreover, screening methods for cutaneous therapeutics require accurate, unsupervised and high-throughput image analysis techniques. RESULTS: By analyzing spectra of images post Gabor filtering and Fast Fourier Transform, we were able to measure subtle changes in collagen fibre orientation intractable to existing techniques. We detected the progressive loss of collagen basketweave structure in a series of chronologically aged skin samples, as well as in skin derived from a model of type 2 diabetes mellitus. CONCLUSIONS: We describe a novel bioimaging approach with implications for the evaluation of pathology in a broader range of biological situations.