Immunoglobulin A (IgA) is generally considered as a non-inflammatory regulator of mucosal immunity, and its importance in diversifying the gut microbiota is increasingly appreciated. IgA autoantibodies have been found in several autoimmune or chronic inflammatory diseases, but their role in pathophysiology is ill-understood. IgA can interact with the Fc receptor FcαRI on immune cells. We now established a novel IgA autoimmune blistering model, which closely resembles the human disease linear IgA bullous disease (LABD) by using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that presence of IgA anti-collagen XVII, - the auto-antigen in LABD-, resulted in neutrophil activation and extravasation from blood vessels into skin tissue. Continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented disease, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage.

BackgroundAs a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs.New methodWe evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery.ResultsWhile the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortexComparison with existing methodsCompared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible.ConclusionsThe study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

Of the adeno-associated viruses (AAVs), AAV9 is known for its capability to cross the blood-brain barrier (BBB) and can, therefore, be used as a noninvasive method to target the central nervous system. Furthermore, the addition of the peptide PhP.B to AAV9 increases its transduction across the BBB by 40-fold. Another neurotropic serotype, AAV5, has been shown as a gene therapeutic delivery vehicle to ameliorate several neurodegenerative diseases in preclinical models, but its administration requires invasive surgery. In this study, AAV9-PhP.B and AAV5-PhP.B were designed and produced in an insect cell-based system. To AAV9, the PhP.B peptide TLAVPFK was added, whereas in AAV5-PhP.B (AQTLAVPFKAQAQ), with AQ-AQAQ sequences used to swap with the corresponding sequence of AAV5. The addition of PhP.B to AAV5 did not affect its capacity to cross the mouse BBB, while increased transduction of liver tissue was observed. Then, intravenous (IV) and intrastriatal (IStr) delivery of AAV9-PhP.B and AAV5 were compared. For AAV9-PhP.B, similar transduction and expression levels were achieved in the striatum and cortex, irrespective of the delivery method used. IStr administration of AAV5 resulted in significantly higher amounts of vector DNA and therapeutic miRNA in the target regions such as striatum and cortex when compared with an IV administration of AAV9-PhP.B. These results illustrate the challenge in developing a vector that can be delivered noninvasively while achieving a transduction level similar to that of direct administration of AAV5. Thus, for therapeutic miRNA delivery with high local expression requirements, intraparenchymal delivery of AAV5 is preferred, whereas a humanized AAV9-PhP.B may be useful when widespread brain (and peripheral) transduction is needed.

Various administration routes of adeno-associated virus (AAV)-based gene therapy have been examined to target the central nervous system to answer the question what the most optimal delivery route is for treatment of the brain with certain indications. In this study, we evaluated AAV5 vector system for its capability to target the central nervous system via intrastriatal, intrathalamic or intracerebroventricular delivery routes in rats. AAV5 is an ideal candidate for gene therapy because of its relatively low level of existing neutralizing antibodies compared to other serotypes, and its broad tissue and cell tropism. Intrastriatal administration of AAV5-GFP resulted in centralized localized vector distribution and expression in the frontal part of the brain. Intrathalamic injection showed transduction and gradient expression from the rostral brain into lumbar spinal cord, while intracerebroventricular administration led to a more evenly, albeit relatively superficially distributed, transduction and expression throughout the central nervous system. To visualize the differences between localized and intra-cerebral spinal fluid administration routes, we compared intrastriatal to intracerebroventricular and intrathecal administration of AAV5-GFP. Together, our results demonstrate that for efficient transgene expression, various administration routes can be applied.

Surgical resection of the primary tumor provides the best chance of cure for patients with colorectal carcinoma (CRC). However, bacterial translocation during intestinal surgery has been correlated with poor long-term oncological outcome. Therefore, we investigated the influence of bacterial contamination during colon surgery on CRC liver metastases development. Blood and liver samples of patients undergoing resection of primary CRC or liver metastases were collected. Cell numbers, activation markers and inflammatory mediators were determined. Tumor cell adhesion and outgrowth after sham- or colectomy operations were determined in a rat model, in which tumor cells had been injected into the portal vein. White blood cells and granulocytes were increased in per- and post-operative patient blood samples. IL-6 was also increased post-operatively compared to the preoperative level. Expression of NOX-2, NOX-4 and polymorphonuclear cells (PMNs) numbers were elevated in post-operative human liver samples. In vitro stimulation of macrophages with plasma of rats after colectomy resulted in production of reactive oxygen species (ROS). Colectomy in rats increased D-lactate levels in plasma, supporting bacterial translocation. Decreased expression of tight junction molecules and increased tumor cell adhesion and outgrowth was observed. Treatment with a selective decontamination of the digestive tract (SDD) cocktail decreased tumor cell adherence after colectomy. In conclusion, postoperative bacterial translocation may activate liver macrophages and PMNs, resulting in ROS production. As we previously showed that ROS release led to liver vasculature damage, circulating tumor cells may adhere to exposed extracellular matrix and grow out into liver metastases. This knowledge is pivotal for development of therapeutic strategies to prevent surgery-induced liver metastases development.

Background Dysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease (CKD). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD, not only by direct effects on the endothelium but also by an increment of α‐Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia‐induced endothelial damage and the extent to which this was dependent on increased α‐Klotho concentrations. Methods and Results In a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial‐gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α‐Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real‐time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro‐permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial‐cadherin–based cell‐cell junctions and diminished F‐actin stress fiber organization, preventing the formation of endothelial intracellular gaps. Conclusions Our results demonstrate that paricalcitol attenuates the CKD‐induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell‐cell interactions.

Immunoglobulin A (IgA) is generally considered a non-inflammatory regulator of mucosal immunity. Conversely, we previously demonstrated that IgA is a potent proinflammatory stimulus for neutrophils after crosslinking of the Fc receptor FcαRI in vitro. The role of FcαRI in IgA-mediated diseases is, however, poorly understood, mostly due to the lack of suitable mouse models. We now established a novel linear IgA bullous disease (LABD) mouse model using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that injection of anti-collagen XVII IgA, which are also present in patients, resulted in neutrophil activation and extravasation from the blood vessels into skin tissue. Moreover, continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented thickening of ear tissue as measure of inflammation, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage due to unrestrained neutrophil activation in patients.

Scar formation is a frequently occurring unwanted result of healing of a skin wound. The mechanisms causing scar formation are not fully understood, although many studies have attempted to unravel this issue. Most of these studies were performed in animals or in in vitro models, but both have major drawbacks. In the present study we aimed to develop a representative model in which the human wound healing process can be studied in vivo. Ex vivo human skin was transplanted it on severely immunocompromized mice. After 90 days, human skin was successfully transplanted on the backs of NSG mice. However, most of the transplants still contained a crust, and the transplant that had shed the crust contracted more than 60% of the original size. Because of the crust and contraction, these transplants are not usable for subsequent wound healing studies yet. In conclusion, we have shown that human skin can successfully be transplanted on NSG mice. In order to be useful, parameters such as skin thickness, or transplantation size could be adapted for improvement of this model.

ABStrACt Ulcerative colitis is a state of chronic inflammation in the colon with unknown etiology, but characterized by massive influx of neutrophils in the inflamed tissue. It is furthermore hypothesized that aberrant T cell responses against commensal microorganisms play a role in the disease, which might be due to disruption of the epithelial barrier function. Immunoglobulin A (IgA), which is ample present in mucosal tissue, can bind to antigens that are present in the lamina propria, leading to the formation of IgA immune complexes. Subsequently, neutrophils are activated through the IgA Fc receptor FcaRI, hereby promoting immune responses. We investigated the role of IgA and FcaRI in the onset of inflammation by inducing experimental colitis in mice that either express human IgA (hIgA) or co-express human IgA and FcaRI (hIgA/FcaRI). The presence of FcaRI aggravated colitis, as reflected by decreased body weight of hIgA/FcaRI mice. Furthermore, increased expression of the Th17 cell-associated cytokines and chemokines CCL2, interleukin (IL)-1b, and IL-6 mRNA expression was observed in intestinal tissues from hIgA/FcaRI mice, which coincided with increased CD4 + T cell numbers. Enhanced release of pro-inflammatory cytokines and chemokines was also observed after cross-linking of FcaRI on human neutrophils. Thus, FcaRI on neutrophils likely plays a role in the induction of a Th17 cell response in experimental colitis.

Immunoglobulin A (IgA) represents the most prominent antibody class at mucosal surfaces and has an important role in mucosal immunity. However, we recently demonstrated in an ex vivo human skin model that cross-linking of the neutrophil IgA Fc receptor (FcaRI) by IgA autoantibodies of linear IgA bullous disease (LABD) patients induced deranged neutrophil recruitment, resulting in tissue damage. LABD is a chronic autoimmune skin blistering disorder characterised by anti-collagen XVII IgA autoantibodies and large neutrophil infiltrates. In the present study, we developed a novel LABD-mouse model to investigate the role of FcaRI in IgA-induced tissue damage in vivo. Therefore, we generated a hybridoma producing human IgA (hIgA) monoclonal antibodies (mAb) directed against mouse collagen XVII (mCOL17). When cryosections of mouse ears were incubated with these mAbs, binding to the basement membrane of the skin was observed. To analyze the role of FcaRI in IgA-induced neutrophil migration in vivo, FcaRI transgenic mice were crossbred with mice containing neutrophils expressing LysEGFP. When anti-mCOL17 hIgA mAbs were injected in the ears of these mice, intravital imaging demonstrated rolling and extravasating neutrophils, which was not observed when human serum IgA was injected as control. Staining cryosections of mouse ears with the granulocyte marker GR-1 confirmed the presence of a large neutrophil infiltrate in response to anti-mCOL17 hIgA, which was not found in cryosections from ears of non-transgenic littermates. Importantly, no rolling or extravasation of neutrophils was observed when mice were injected with an FcaRI blocking antibody. This indicates that IgA-induced neutrophil activation and migration is dependent on FcaRI in vitro and in vivo. Thus, developing agents that specifically block IgA-FcaRI interactions may represent a promising novel approach for IgA-induced blistering diseases.

Sézary syndrome ( SS ) is an aggressive cutaneous T ‐cell lymphoma with CD 4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG 2 −/− γc −/− mice that are completely devoid of T‐, B‐ and NK ‐cell activity. Injection of the SS cell line SeAx led to long‐term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT‐78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD 3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG 2 −/− γc −/− mice results in long‐term systemic repopulation of the mice, thereby providing a novel mouse model for S ézary syndrome.

Surgical resection of the primary tumor provides the best chance of cure for patients with colorectal cancer (CRC). However, we previously demonstrated that laparotomy led to enhanced adhesion of tumor cells in the liver vasculature. Moreover, it was demonstrated that patients with anastomotic leakage after resection of the tumor had poorer survival, supporting that resection of primary CRC paradoxically may have a negative impact on metastases development and long-term patient outcome. Therefore, the aim of this study was to investigate the influence of bacterial contamination during surgery on liver metastases development. Rats underwent a sham operation (laparotomy, opening and closure of peritoneal cavity), colectomy (resection of part of the colon and anastomosis), or received anesthesia alone, after which tumor cells were injected into the portal circulation. When swaps of the colon wall were taken at the beginning of surgery, no bacterial outgrowth was observed. However, an increased number of bacterial colonies grew out when swaps were taken of the suture, supporting bacterial contamination after subtotal colectomy. Moreover, both tumor cell adhesion and liver metastases development were significantly enhanced in rats that underwent colectomy. Additionally, liver vessel integrity was disrupted, as expression of the tight junction molecules ZO-1 and Claudin-5 were decreased after laparotomy and subtotal colectomy, indicating loss of cell-cell contact after abdominal surgery. This was likely due to increased macrophage activity. In conclusion, our results support that macrophages were activated by a systemic factor in the plasma of operated rats, which led to impaired liver vasculature and augmented tumor cells adhesion and outgrowth. Exposure to bacterial products after colectomy may contribute to this process, as this will strongly activate macrophages. Understanding the precise mechanisms may aid the rational design of novel strategies to prevent liver metastases development after bacterial contamination, hereby improving prognosis of patients undergoing CRC resection. 4 COLECTOMY ENHANCES TUMOR CELL ADHESION

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2(-/-)IL-2Rγc(-/-) mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRPα; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRPα, we introduced functional CD47/SIRPα interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T- and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68- and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRPα interactions, human T cells accumulated as CD4(+) or CD8(+) single-positive, naive, resting T cells in the presence of functional CD47/SIRPα interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRPα is a critical determinant of T- and NK-cell homeostasis under steady-state conditions in vivo.

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated ‘human immune system’ (HIS) mice by transplanting newborn BALB/c Rag2−/−IL-2Rγc−/− immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

Over the last two decades, several humanized mouse models have been used to experimentally analyze the function and development of the human immune system. Recent advances have lead to the establishment of new murine-human chimeric models with improved characteristics, both in terms of human engraftment efficiency and in situ multilineage human hematopoietic development. We describe here the use of newborn BALB/c Rag2(-/-)gamma(c) (-/-) mice as recipients of human hematopoietic progenitor cells to produce "human immune system" (HIS) (BALB-Rag/gamma) mice, using human fetal liver progenitors. The two major subsets of the human dendritic cell lineage, namely, BDCA2(+)CD11c(-) plasmacytoid dendritic cells and BDCA2(-)CD11c(+) conventional dendritic cells, can be found in HIS (BALB-Rag/gamma) mice. In order to manipulate the expression of genes of interest, the human hematopoietic progenitor cells can be genetically engineered ex vivo by lentiviral transduction before performing xenograft transplantation. Using this mouse model, the human immune system can be assessed for both fundamental and pre-clinical purposes.