Accurate determination of tumor human epidermal growth factor receptor 2 (HER2)-status in breast cancer patients is possible via noninvasive imaging, provided adequate tracers are used. In this study, we describe the generation of a panel of 38 nanobodies, small HER2-binding fragments that are derived from heavy-chain-only antibodies raised in an immunized dromedary. In search of a lead compound, a subset of nanobodies was biochemically characterized in depth and preclinically tested for use as tracers for imaging of xenografted tumors. The selected compound, 2Rs15d, was found to be stable and to interact specifically with HER2 recombinant protein and HER2-expressing cells in ELISA, surface plasmon resonance, flow cytometry, and radioligand binding studies with low nanomolar affinities, and did not compete with anti-HER2 therapeutic antibodies trastuzumab and pertuzumab. Single-photon-emission computed tomography (SPECT) imaging quantification and biodistribution analyses showed that (99m)Tc-labeled 2Rs15d has a high tumor uptake in 2 HER2(+) tumor models, fast blood clearance, low accumulation in nontarget organs except kidneys, and high concomitant tumor-to-blood and tumor-to-muscle ratios at 1 h after intravenous injection. These values were dramatically lower for an irrelevant control (99m)Tc-nanobody and for (99m)Tc-2Rs15d targeting a HER2(-) tumor.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (M s)were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident M types, designated MHC-II lo and MHC-II hi M s, the latter being predominant. MHC-II lo and MHC-II hi pancreas M s differed at the molecular level, with MHC-II lo M s being more M2-activated. After PDL, there was an early surge of Ly6C hi monocyte infiltration in the pancreas, followed by a transient MHC-II lo M peak and ultimately a restoration of the MHC-II hi M -dominated steadystate equilibrium. These intricate M dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local M proliferation. Functionally, MHC-II lo M s were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue M s, rather than Ly6C hi monocyte-derived M s, contributed to β-cell proliferation. Together, our study fully characterizes the M subsets in the pancreas and clarifies the complex dynamics of M s after PDL injury.Keywords: M activation r M heterogeneity r M proliferation r Pancreas inflammation Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Prof. Harry Heimberg and Prof. Jo A. Van Ginderachter e-mail: harry.heimberg@vub.ac.be e-mail: jvangind@vub.ac.be * These authors contributed equally to this study as first co-authors. * * These authors contributed equally to this study as senior co-authors.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 1482-1493 Innate immunity 1483Introduction M s display a tremendous plasticity in vivo depending on their microenvironment. Their activation status has been described using the conceptual framework of M1 (classical) and M2 (alternative) activation [1], although this model oversimplifies their true plasticity. In addition to their role as immune cells, the trophic role of M s during development, tissue repair, and regeneration is becoming increasingly appreciated [1,2]. Hence, efforts are being made to dissect specialized M subpopulations and to trace their origin in situations of sterile inflammation. Although tissueresident M s were traditionally believed to originate from bloodborne monocytes, recent evidence shows that such M s develop prenatally from dedicated precursors [3,4]. During adulthood, the pool of tissue-resident M s is maintained by low-level proliferation under steady-state [5] and can expand by enhanced in situ proliferation during pathologies [6][7][8][9]. Moreover, during sterile inflammat...

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Macrophages are classically considered detrimental for pancreatic b-cell survival and function, thereby contributing to b-cell failure in both type 1 (T1D) and 2 (T2D) diabetes mellitus. In addition, adipose tissue macrophages negatively influence peripheral insulin signaling and promote obesityinduced insulin resistance in T2D. In contrast, recent data unexpectedly uncovered that macrophages are not only able to protect b cells during pancreatitis but also to orchestrate b-cell proliferation and regeneration after b-cell injury. Moreover, by altering their activation state, macrophages are able to improve insulin resistance in murine models of T2D. This review will elaborate on current insights in macrophage heterogeneity and on the evolving role of pancreas macrophages during organogenesis, tissue injury, and repair. Additional identification of macrophage subtypes and of their secreted factors might ultimately translate into novel therapeutic strategies for both T1D and T2D. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:555-563 SIGNIFICANCEDiabetes mellitus is a pandemic disease, characterized by severe acute and chronic complications. Macrophages have long been considered prime suspects in the pathogenesis of both type 1 and 2 diabetes mellitus. In this concise review, current insights in macrophage heterogeneity and on the, as yet, underappreciated role of alternatively activated macrophages in insulin sensing and b-cell development/repair are reported. Further identification of macrophage subtypes and of their secreted factors might ultimately translate into novel therapeutic strategies for diabetes mellitus.

Aims/hypothesis Vascular endothelial growth factor (VEGF) has been recognised by loss-of-function experiments as a pleiotropic factor with importance in embryonic pancreas development and postnatal beta cell function. Chronic, nonconditional overexpression of VEGF-A has a deleterious effect on beta cell development and function. We report, for the first time, a conditional gain-of-function study to evaluate the effect of transient VEGF-A overexpression by adult pancreatic beta cells on islet vasculature and beta cell proliferation and survival, under both normal physiological and injury conditions.

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycaemic alterations and life-threatening complications. Restoration of the endogenous β-cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive β-cell regeneration remain enigmatic and β-cell replacement therapy still faces major hurdles that prevent its widespread application. Co-transplantation of accessory non-islet cells with islet cells has been shown to improve the outcome of experimental islet transplantation. This review will highlight current travails in β-cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes.

Reduced intra-islet VEGF-A signalling results in maternal islet hypovascularisation and impaired glycaemic control but does not preclude beta cell expansion during mouse pregnancy.