Animal models for basic and translational research in reconstructive transplantation

Reconstructive transplantation represents a bona fide option for select patients with devastating tissue loss, which could better restore the appearance, anatomy, and function than any other conventional treatment currently available. Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the potential side effects of chronic multidrug immunosuppression. Thus, any reconstructive measures to improve these non‐life‐threatening conditions must address a delicate balance of risks and benefits. Today, several exciting novel therapeutic strategies, such as the implementation of cellular therapies including bone marrow or stem cells that integrate the concepts of immune regulation with those of nerve regeneration, are on the horizon. The development of reliable and reproducible small andlarge animal models is essential for the study of the unique immunological and biological aspects of vascularized composite allografts and to translate such novel immunoregulatory and tolerance‐inducing strategies and therapeutic concepts from the bench to bedside. This review provides an overview of the multitude of small and largeanimal models that have been particularly designed for basicand translational research related to reconstructive transplantation. (Part C) 96:39–50, 2012. © 2012 Wiley Periodicals, Inc.     

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.     

Applicability of bioengineered human skin: from preclinical skin humanized mouse models to clinical regenerative therapies

Ongoing progress in the field of regenerative medicine, in combination with the development of tissue-engineered skin products, has opened new possibilities for the treatment of certain diseases in which current treatments are aimed at alleviating symptoms but are not able to get a permanent cure. Our laboratory has developed a fibrin-based bioengineered human skin that has been successfully used for permanent regenerative therapies in different situations in the clinic. Moreover, we have been able to stably regenerate human skin by orthotopic grafting of this skin equivalent onto the back of immunodeficient mice. The so-called skin-humanized mouse model system has permitted us to model several monogenic skin diseases, when keratinocytes and fibroblasts harboring the genetic defect were used. In most cases different gene therapy approaches for ex vivo correction of cells have proved effective in reverting the phenotype using this model. More importantly, the feasibility of the system has allowed us to generate a skin humanized mouse model for psoriasis, a common chronic inflammatory disease where the immune component has a pivotal role in the pathogenesis. Establishing reliable humanized animal models for skin diseases is necessary to gain a deeper knowledge of the pathogenesis and to develop novel therapeutic strategies. In this sense, the skin humanized mouse model developed in our laboratory meets the needs of this field of research.     

Epigenetic regulation of immune cell functions during post-septic immunosuppression

Studies in humans and animal models indicate that profound immunosuppression is one of the chronic consequences of severe sepsis. This immune dysfunction encompasses deficiencies in activation of cells in both the myeloid and lymphoid cell lineages. As a result, survivors of severe sepsis are at risk of succumbing to infections perpetrated by opportunistic pathogens that are normally controlled by a fully functioning immune system. Recent studies have indicated that epigenetic mechanisms may be one driving force behind this immunosuppression, through suppression of proinflammatory gene production and subsequent immune cell activation, proliferation and effector function. A better understanding of epigenetics and post-septic immunosuppression can improve our diagnostic tools and may be an important potential source of novel molecular targets for new therapies. This review will discuss important pathways of immune cell activation affected by severe sepsis, and highlight pathways of epigenetic regulation that may be involved in post-septic immunosuppression.     

Epigenetic regulation of immune cell functions during post-septic immunosuppression

Studies in humans and animal models indicate that profound immunosuppression is one of the chronic consequences of severe sepsis. This immune dysfunction encompasses deficiencies in activation of cells in both the myeloid and lymphoid cell lineages. As a result, survivors of severe sepsis are at risk of succumbing to infections perpetrated by opportunistic pathogens that are normally controlled by a fully functioning immune system. Recent studies have indicated that epigenetic mechanisms may be one driving force behind this immunosuppression, through suppression of proinflammatory gene production and subsequent immune cell activation, proliferation and effector function. A better understanding of epigenetics and post-septic immunosuppression can improve our diagnostic tools and may be an important potential source of novel molecular targets for new therapies. This review will discuss important pathways of immune cell activation affected by severe sepsis, and highlight pathways of epigenetic regulation that may be involved in post-septic immunosuppression.Key words: sepsis, immunosuppression, histone modification, gene regulation, inflammation, macrophages, dendritic cells, T lymphocytes     

Nitric oxide-mediated depletion of Langerhans cells from the epidermis may be involved in UVA radiation-induced immunosuppression.

Ultraviolet A (UVA) irradiation of the dorsal skin of mice reduced the contact hypersensitivity (CHS) response and the density of epidermal Langerhans cells (LC). The roles of nitric oxide (NO) and reactive oxygen species (ROS) in these biological effects of UVA were investigated. Topical application of N(G)-monomethyl-L-arginine acetate, an inhibitor of NO production, 2,2'-dipyridyl, an iron chelater, or 4-hydroxy-tempo, a superoxide dismutase mimicking agent, inhibited UVA-induced suppression of the CHS response. N(G)-monomethyl-L-arginine acetate but not the ROS inhibitors prevented UVA from reducing LC numbers in the epidermis. This suggests that NO but not ROS produced in response to UVA mediates a depletion of LC from the epidermis, probably by signaling these cells to migrate from the skin. This could be responsible for UVA-induced immunosuppression. UVA-induced ROS can also cause immunosuppression, but by a different mechanism. Agents that inhibit or modulate NO or ROS production may be useful for preventing damage caused by the UVA component of sunlight to the skin immune system.     

DNA damage initiates photobiologic reactions in the skin.

Ultraviolet (UV) radiation can induce acute and chronic photobiologic reactions in the absence of exogenous chromophores. Nuclear DNA is a major chromophore to initiate UV-induced physiologic reactions. The XPA-gene deficient mouse, an animal model of xeroderma pigmentosum, develops increased photobiologic reactions including acute inflammation, immunosuppression and skin cancers, because of the defect in the excision repair of UV-induced DNA lesions.     

Short-term and long-term cellular and molecular events following UV irradiation of skin: implications for molecular medicine

Acute ultraviolet (UV) irradiation of normal human skin results in several clinical effects, including sunburn inflammation (erythema) and tanning, histological changes such as thickening of the epidermis, and local or systemic immunosuppression. Chronic UV irradiation leads to photoaging, sustained immunosuppression and photocarcinogenesis. Photocarcinogenesis involves the accumulation of genetic changes, as well as immune system modulation, and ultimately leads to the development of skin cancers. Recent advances in molecular and cellular biology have clarified the mechanisms of photocarcinogenesis, including the formation of DNA photoproducts, DNA repair, the mutation of proto-oncogenes and tumour suppressor genes, and UV-induced immunosuppression. Further investigation and a better understanding of photocarcinogenesis are critical to the development of effective prevention and intervention strategies for human skin cancer.     

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.     

Development of highly organized lymphoid structures in Buruli ulcer lesions after treatment with rifampicin and streptomycin

Background

Buruli ulcer caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic necrotising skin ulcers. The pathogenesis is associated with the cytocidal and immunosuppressive activities of a macrolide toxin. Histopathological hallmark of progressing disease is a poor inflammatory response despite of clusters of extracellular bacilli. While traditionally wide excision of the infected tissue was the standard treatment, provisional WHO guidelines now recommend an eight week pre-treatment with streptomycin and rifampicin.

Methodology/Principal Findings

We conducted a detailed immunohistochemical analysis of tissue samples from Buruli patients who received antibiotic treatment. Cellular immune response along with bacterial load and distribution were monitored. We demonstrate that this treatment leads to the development of highly organized cellular infiltration surrounding areas of coagulative necrosis. Diffuse infiltrates, granulomas and dense lymphocyte aggregation close to vessels were observed. Mycobacterial material was primarily located inside mononuclear phagocytes and microcolonies consisting of extracellular rod-shaped mycobacteria were no longer found. In observational studies some patients showed no clinical response to antibiotic treatment. Corresponding to that, one of five lesions analysed presented with huge clusters of rod-shaped bacilli but no signs of infiltration.

Conclusions/Significance

Results signify that eight weeks of antibiotic treatment reverses local immunosuppression and leads to an active inflammatory process in different compartments of the skin. Structured leukocyte infiltrates with unique signatures indicative for healing processes developed at the margins of the lesions. It remains to be analysed whether antibiotic resistance of certain strains of M. ulcerans, lacking patient compliance or poor drug quality are responsible for the absent clinical responses in some patients. In future, analysis of local immune responses could serve as a suitable surrogate marker for the efficacy of alternative treatment strategies.     

Immune Profiles to Predict Response to Desensitization Therapy in Highly HLA-Sensitized Kidney Transplant Candidates

BackgroundKidney transplantation is the most effective treatment for end-stage kidney disease. Sensitization, the formation of human leukocyte antigen (HLA) antibodies, remains a major barrier to successful kidney transplantation. Despite the implementation of desensitization strategies, many candidates fail to respond. Current progress is hindered by the lack of biomarkers to predict response and to guide therapy. Our objective was to determine whether differences in immune and gene profiles may help identify which candidates will respond to desensitization therapy.ConclusionsMeasuring baseline and longitudinal immune and gene profiles could provide a useful strategy to distinguish responders from non-responders to desensitization therapy. This study presents the integration of novel translational studies including CyTOF immunophenotyping in a multivariate analysis model that has potential applications to predict response to desensitization, select candidates, and personalize medicine to ultimately improve overall outcomes in highly sensitized kidney transplant candidates.     

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.     

Neurobiology of comorbid post‐traumatic stress disorder and alcohol‐use disorder

Post‐traumatic stress disorder (PTSD) and alcohol‐use disorder (AUD) are highly comorbid in humans. Although we have some understanding of the structural and functional brain changes that define each of these disorders, and how those changes contribute to the behavioral symptoms that define them, little is known about the neurobiology of comorbid PTSD and AUD, which may be due in part to a scarcity of adequate animal models for examining this research question. The goal of this review is to summarize the current state‐of‐the‐science on comorbid PTSD and AUD. We summarize epidemiological data documenting the prevalence of this comorbidity, review what is known about the potential neurobiological basis for the frequent co‐occurrence of PTSD and AUD and discuss successes and failures of past and current treatment strategies. We also review animal models that aim to examine comorbid PTSD and AUD, highlighting where the models parallel the human condition, and we discuss the strengths and weaknesses of each model. We conclude by discussing key gaps in our knowledge and strategies for addressing them: in particular, we (1) highlight the need for better animal models of the comorbid condition and better clinical trial design, (2) emphasize the need for examination of subpopulation effects and individual differences and (3) urge cross‐talk between basic and clinical researchers that is reflected in collaborative work with forward and reverse translational impact.     

KM突变小鼠慢性炎症性皮肤病的免疫学改变

目的 观察KM突变小鼠皮肤慢性炎症的病理变化,探讨该小鼠皮肤免疫学改变.方法 通过外部特征、常规HE病理组织学、免疫组织化学、特染方法对3月龄、6月龄KM突变小鼠皮肤炎症细胞及炎症因子进行检测并与KM野生小鼠皮肤炎症细胞及炎症因子浸润进行比较.结果 KM突变小鼠皮肤毛稀、皮屑、皮皱等;组织病理表皮细胞坏死,上皮角化过度或不全,颗粒层增厚,基底细胞层水肿,真皮浅层血管扩张,结缔组织炎细胞浸润等,皮肤CD3+、CD4+T细胞、巨噬细胞、肥大细胞等增多,同时炎症因子IL-6、IL-22、TNF-α、IFN-γ等增多;且这些炎症细胞及炎症因子浸润3月龄较6月龄增多.结论 KM自发突变小鼠皮肤组织出现自发的慢性炎症病变,与人类慢性炎症性皮肤病变有相类似的病理改变和细胞分子改变,有望培育成为一种新的慢性炎症性皮肤病的动物模型.     

Global remodelling of cellular microenvironment due to loss of collagen VII

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post‐translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF‐β and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.     

Translational cognitive neuroscience of dementia with touchscreen operant chambers

Translational cognitive neuroscience of dementia involves mainly two areas: the validation of newly developed dementia animal models and the preclinical assessment of novel drug candidates in such model animals. To validate new animal models, a multidomain panel (battery) approach is essential in that dementia is, by definition, not merely a memory disorder but rather a multidomain cognitive/behavior disorder: animal modeling with a certain type of dementia would develop cognitive impairments in multiple (two at minimum) domains in a specific order according to unique spreading patterns of its neuropathology. In new drug development, the availability of highly sensitive tools assessing animal cognition is crucial to the detection of cognitive decline at the earliest stage of the disease, which may be an optimal time point to test a drug candidate. Using interspecies translatable (analogous) cognitive tasks would also be necessary to successfully predict the efficacy of drug candidates in subsequent clinical trials. Currently, this translational prediction is seriously limited given discrepancies in behavioral assessment methods between animals and humans in the preclinical and clinical trials, respectively. Since neurodegenerative diseases are often accompanied by not only cognitive but also affective and movement disorders, simultaneous assessment of task-relevant locomotor behavior and motivation is also important to rule out the effects of potential confounders. The touchscreen operant platform may satisfy these needs by offering several advantages over conventional methodology. In this review, we discuss the touchscreen operant chamber system and highlight some of its qualities as a promising and desirable tool for translational research of dementia.     

Adhesion molecules: Key players in Mesenchymal stem cell-mediated immunosuppression

Adhesion molecules are known to -be important components of an active T-cell mediated immune response. Signals generated at a site of inflammation cause circulating T cells to respond by rolling, arrest and then transmigration through the endothelium, all of which are mediated by adhesion molecules. Consequently, strategies have been developed to treat immune disorders with specific antibodies that block the interaction of adhesion molecules. However, the therapeutic effects of such remedies are not always achieved. Our recent investigations have revealed that intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) work together with chemokines to induce immunosuppression mediated by Mesenchymal stem cells (MSCs), thus demonstrating the dual role of adhesion molecules in immune responses. Since MSCs represent an important component of the stromal cells in an inflammatory microenvironment, our findings provide novel information for understanding the regulation of immune responses and for designing new strategies to treat immune disorders.Adhesion molecules are cell surface proteins that mediate the interaction between cells, or between cells and the extracellular matrix (ECM). There are four families of adhesion molecules: immunoglobulin-like adhesion molecules, integrins, cadherins and selectins. Most of them are typical transmembrane proteins that have cytoplasmic, transmembrane and extracellular domains. In the immune system, cell adhesion plays a critical role in initiating and sustaining an effective immune response against foreign pathogens.¹ Based on our recent data, we discuss herein the role of immunoglobulin superfamily cell adhesion molecules, ICAM1 and VCAM-1, in the immunosuppression mediated by Mesenchymal stem cells.     

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.     

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

Macrophages are key players in controlling the immune response that can adapt to microenvironmental signals. This results in distinct polarization states (classical M1 or alternative M2), that play a differential role in immune regulation. In general, the M1 contribute to onset of inflammation, whereas the M2 orchestrate resolution and repair, whereby failure to switch from predominantly M1 to M2 reinforces a pro-inflammatory environment and chronic inflammation. Here, we show selective elimination of M1 macrophages in vitro by a range of CD64-targeted immunotoxins, including H22(scFv)-ETA'. After re-polarization of already polarized macrophages, still only M1 polarization showed sensitivity toward CD64-directed immunotoxins. The selectivity for M1 was found linked to reduced endosomal protease activity in M1 macrophages as demonstrated by inhibition of endosomal proteases. Using the H22(scFv)-ETA' in a transgenic mouse model for chronic cutaneous inflammation, the M1 specificity was confirmed in vivo and a beneficial effect on inflammation demonstrated. Also ex vivo on skin biopsies from atopic dermatitis and diabetes type II patients with chronically-inflamed skin, a clear M1 specific effect was found. This indicates the potential relevance for human application. Our data show that targeting M1 macrophages through CD64 can be instrumental in developing novel intervention strategies for chronic inflammatory conditions.