1,25-Dihydroxycholecalciferol with low-calcium diet reduces acute rejection in rat lung allotransplantation

The effect of 1,25-dihydroxycholecalciferol (calcitriol, vitamin D3) with a low-calcium diet on the acute lung allograft rejection in a rat unilateral left lung transplantation model was evaluated.

Continuous flow left ventricular assist devices: a valid option for heart failure patients

Recent outstanding clinical advances with new mechanical circulatory systems (MCS) have led to additional strategies in the treatment of end stage heart failure (HF). Heart transplantation (HTx) can be postponed and for certain patients even replaced by smaller implantable left ventricular assist devices (LVAD). Mechanical support of the failing left ventricle enables appropriate hemodynamic stabilisation and recovery of secondary organ failure, often seen in these severely ill patients. These new devices may be of great help to bridge patients until a suitable cardiac allograft is available but are also discussed as definitive treatment for patients who do not qualify for transplantation. Main indications for LVAD implantation are bridge to recovery, bridge to transplantation or destination therapy. LVAD may be an important tool for patients with an expected prolonged period on the waiting list, for instance those with blood group 0 or B, with a body weight over 90 kg and those with potentially reversible secondary organ failure and pulmonary artery hypertension. However, LVAD implantation means an additional heart operation with inherent peri-operative risks and complications during the waiting period. Finally, cardiac transplantation in patients with prior implantation of a LVAD represents a surgical challenge. This review summarises the current knowledge about LVAD and continuous flow devices especially since the latter have been increasingly used worldwide in the most recent years. The review is also based on the institutional experience at Berne University Hospital between 2000 and 2012. Apart from short-term devices (Impella, Cardiac Assist, Deltastream and ECMO) which were used in approximately 150 cases, 85 pulsatile long-term LVAD, RVAD or bi-VAD and 44 non-pulsatile LVAD (mainly HeartMateII and HeartWare) were implanted. After an initial learning curve, one-year mortality dropped to 10.4% in the last 58 patients.

Mycobacterium microti infection in the cat: a case report, literature review and recent clinical experience

OVERVIEW: Mycobacterium microti infection is infrequently described in cats in the veterinary literature. It can be one of a large number of possible differential diagnoses in a feline patient with dermal nodules and non-healing draining ulcers, and can occasionally spread to involve the lungs and/or other areas of the body. CASE SUMMARY: This report describes the clinical signs, eventual diagnosis and variable response to treatment in a cat in Switzerland with recurrent cutaneous M microti infection. Only after several diagnostic and therapeutic attempts, over more than 2 years, was the species of Mycobacterium finally identified and targeted therapy given. PRACTICAL RELEVANCE: For any cat in which there is even a low suspicion of mycobacterial infection, the authors recommend that an aggressive diagnostic approach is taken. Tissue specimens should be collected and frozen early on, and, as soon as acid-fast bacilli are detected, samples should be sent to a mycobacterial reference laboratory for definitive identification. LITERATURE REVIEW: A review of the literature relating to the aetiopathogenesis, diagnosis and management of M microti infection in cats and dogs is included. This is supplemented with clinical and therapeutic experience gained from this case and other, unpublished cases managed over the past 15 years by one of the authors (DGM).

New drug targets in atopic dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by eczematous skin lesions, pruritus and typical histopathological features. T cells are thought to play a key role, but B cells might also participate in the pathogenesis of AD. In two investigator-initiated pilot studies, we studied the effects of B cell depletion by monoclonal anti-CD20 antibody therapy or a reduction of activated T cells by LFA3-IgG fusion protein on moderate-to-severe AD. All patients treated with either rituximab or alefacept showed an improvement of their skin symptoms with a sustained effect after treatment. In both studies, histological alterations, such as spongiosis, acanthosis and dermal infiltrate, including T and B cell numbers, dramatically improved and the expression of IL-5 and IL-13 was reduced after therapy. Upon rituximab therapy, allergen-specific IgE levels were not altered and total serum IgE levels only slightly decreased. According to recent studies, neutralizing B and T cells products such as IgE or IL-5 might be effective in subgroups of patients with AD.

Characterization of canine dendritic cells in healthy, atopic, and non-allergic inflamed skin

Atopic dermatitis in humans and dogs is a chronic relapsing allergic skin disease. Dogs show a spontaneous disease similar to the human counterpart and represent a model to improve our understanding of the immunological mechanisms, the pathogenesis of the disease, and new therapy development. The aim of the study was to determine the frequency and phenotype of dendritic cells (DC) in the epidermis and dermis of healthy, canine atopic dermatitis lesional, and non-allergic inflammatory skin to further validate the model and to obtain insights into the contribution of DC to the pathogenesis of skin diseases in dogs. We first characterized canine skin DC using flow-cytometric analysis of isolated skin DC combined with an immunohistochemical approach. A major population of canine skin dendritic cells was identified as CD1c(+)CD11c(+)CD14(-)CD80(+)MHCII(+)MAC387(-) cells, with dermal DC but not Langerhans cells expressing CD11b. In the epidermis of lesional canine atopic dermatitis and non-allergic inflammatory skin, we found significantly more dendritic cells compared with nonlesional and control skin. Only in canine atopic dermatitis skin did we find a subset of dendritic cells positive for IgE, in the epidermis and the dermis. Under all inflammatory conditions, dermal dendritic cells expressed more CD14 and CD206. MAC387(+) putative macrophages were absent in healthy but present in inflamed skin, in particular during non-allergic diseases. This study permits a phenotypic identification and differentiation of canine skin dendritic cells and has identified markers and changes in dendritic cells and macrophage populations related to allergic and non-allergic inflammatory conditions. Our data suggest the participation of dendritic cells in the pathogenesis of canine atopic dermatitis similar to human atopic dermatitis and further validate the only non-murine spontaneous animal model for this disease.

Evaluation and quantification of spectral information in tissue by confocal microscopy

A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications.

Repeat-dose sirolimus pharmacokinetics and pharmacodynamics in patients with hepatic allografts

To determine sirolimus steady-state pharmacokinetics, and to assess the relationship between time-normalized trough sirolimus concentration (C(min,TN)) and evidence of efficacy (rejection and death) and adverse reactions (stomatitis and pneumonia) in liver allograft patients.

The Effects Of Farnesyltransferase Inhibitor Abt-100 On Murine Helper T-Cell Differentiation And Cytokine Secretion

Farnesyltransferase Inhibitors (FTIs) are a class of drugs known to prevent the farnesylation and subsequent membrane attachment of a number of intracellular proteins. In various studies, the administration of FTIs has been found to play a role in the activation and development of T-cells in the immune system. FTIs have also been found to act as immunomodulators in delaying MHC-II mismatched skin allografts in mice. This study focuses on the effect of the FTI, ABT-100, on the differentiation and cytokine secretion of Th1 and Th2 helper T-cells in BALB/C mice to better understand which immune responses are targeted by FTIs. Splenocytes were isolated from BALB/C mice, skewed towards either a Th1 or a Th2 phenotype with the addition of cytokines, and treated with various concentrations of ABT-100. Splenocytes were also isolated and immediately cultured in the presence of ABT-100 to observe differentiation trends of helper T-cells. Cytokine production was measured using intracytoplasmic flow cytometry analysis. I found that ABT-100 treatment does not block Th1 or Th2 cell differentiation. Instead, ABT-100 treatment appears to affect cytokine production from effector T-cells. I found that ABT-100 causes a decrease in IFN-¿ production in mature Th1 cells yet does not affect IL-4 production in mature Th2 cells. This decrease in cytokine production as a result of ABT-100 treatments provides a potential mechanism for how ABT-100 works to delay MHC-II mismatched allograft rejection.

Endothelial cell antibody-mediated rejection and successful retransplantation in a heart transplanted patient

Antibody-mediated rejection (AMR) plays a significant role in cardiac allograft dysfunction, and recently a consensus regarding the diagnosis of AMR has been published. To our knowledge, it has not previously been reported that acute graft failure related to AMR, and antiendothelial cell antibodies can successfully be diagnosed to allow the patient to receive the outlined treatment and undergo a subsequent retransplantation.

Uninephrectomy reduces 11β-hydroxysteroid dehydrogenase type 1 and type 2 concomitantly with an increase in blood pressure in rats

Renal allograft donors are at risk of developing hypertension. Here, we hypothesized that this risk is at least in part explained by an enhanced intracellular availability of 11β-hydroxyglucocorticoids due to an increased 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1), an intracellular prereceptor activator of biologically inactive 11-ketocorticosteroids in the liver, and/or a diminished 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an inactivator of 11β-hydroxyglucocorticoids in the kidney. To test this hypothesis, uninephrectomized (UNX) (n=9) and sham-operated (n=10) adult Sprague-Dawley rats were investigated. Mean arterial blood pressure and heart rate were measured continuously by telemetry for 6 days in week 5 after UNX. The mRNA of 11β-Hsd1 and 11β-Hsd2 in liver and kidney tissues were assessed by RT-PCR and the 11β-HSD activities were directly quantified in their corresponding tissues by determining the ratios of (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) and of corticosterone/dehydrocorticosterone (B/A) by gas chromatography-mass spectrometry. The apparent total body activities of 11β-HSD1 and 11β-HSD2 were estimated using the urinary and plasma ratios of (THB+5α-THB)/THA and B/A. Mean arterial blood pressure was increased after UNX when compared with sham operation. Hepatic mRNA content of 11β-Hsd1 and hepatic, plasma, and urinary ratios of (THB+5α-THB)/THA were decreased after UNX, indicating diminished access of glucocorticoids to its receptors. In renal tissue, 11β-Hsd2 mRNA was reduced and B/A ratios measured in kidney, plasma, and urine were increased, indicating reduced 11β-HSD2 activity and enhanced access of glucocorticoids to mineralocorticoid receptors. Both 11β-HSD1 and 11β-HSD2 are downregulated after UNX in rats, a constellation considered to induce hypertension.

Evaluation of a porcine whole-limb heterotopic autotransplantation model

Recently performed vascularized composite tissue allotransplantations (CTAs) stimulate the ongoing research in the area of whole-limb transplantation. A reliable in vivo animal model is required for investigations in vascularized whole-limb CTA. The model should allow in vivo assessment in whole-limb preservation, allograft and xenograft response, and host immunomodulation. The goal of this study is to describe and evaluate the in vivo feasibility and reproducibility of a whole-limb porcine model as a basis for future research in this field.

Mycophenolate acid inhibits endothelial NAD(P)H oxidase activity and superoxide formation by a Rac1-dependent mechanism

Endothelial dysfunction precedes hypertension and atherosclerosis and predicts cardiac allograft vasculopathy and death in heart transplant recipients. Endothelial overproduction of reactive oxygen species, such as superoxide anions produced by NAD(P)H oxidase, induces endothelial dysfunction. Because immunosuppressive drugs have been associated with increased reactive oxygen species production and endothelial dysfunction, we sought to elucidate the underlying mechanisms. Reactive oxygen species, release of superoxide anions, and NAD(P)H oxidase activity were studied in human umbilical vein endothelial cells and in polymorphonuclear neutrophils. Gp91ds-tat was used to specifically block NAD(P)H oxidase. Transcriptional activation of different subunits of NAD(P)H oxidase was assessed by real-time RT-PCR. Rac1 subunit translocation and activation were studied by membrane fractionation and pull-down assays. Calcineurin inhibitors significantly increased endothelial superoxide anions production because of NAD(P)H oxidase, whereas mycophenolate acid (MPA) blocked it. MPA also attenuated the respiratory burst induced by neutrophil NAD(P)H oxidase. Because transcriptional activation of NAD(P)H oxidase was not affected, but addition of guanosine restored endothelial superoxide anions formation after MPA treatment, we speculate that the inhibitory effect of MPA was mediated by depletion of cellular guanosine triphosphate content. This prevented activation of Rac1 and, thus, of endothelial NAD(P)H oxidase. Because all heart transplant recipients are at risk for cardiac allograft vasculopathy development, these differential effects of immunosuppressants on endothelial oxidative stress should be considered in the choice of immunosuppressive drugs.

Translation from research to applications

The article summarizes the collective views expressed at the fourth session of the workshop Tissue Engineering-the Next Generation, which was devoted to the translation of results of tissue engineering research into applications. Ernst Hunziker described the paradigm of a dual translational approach, and argued that tissue engineering should be guided by the dimensions and physiological setting of the bodily compartment to be repaired. Myron Spector discussed collagen-glycosaminoglycan (GAG) scaffolds for musculoskeletal tissue engineering. Jeanette Libera focused on the biological and clinical aspects of cartilage tissue engineering, and described a completely autologous procedure for engineering cartilage using the patient's own chondrocytes and blood serum. Arthur Gertzman reviewed the applications of allograft tissues in orthopedic surgery, and outlined the potential of allograft tissues as models for biological and medical studies. Savio Woo discussed a list of functional tissue engineering approaches designed to restore the biochemical and biomechanical properties of injured ligaments and tendons to be closer to that of the normal tissues. Specific examples of using biological scaffolds that have chemoattractants as well as growth factors with unique contact guidance properties to improve their healing process were shown. Anthony Ratcliffe discussed the translation of the results of research into products that are profitable and meet regulatory requirements. Michael Lysaght challenged the proposition that commercial and clinical failures of early tissue engineering products demonstrate a need for more focus on basic research. Arthur Coury described the evolution of tissue engineering products based on the example of Genzyme, and how various definitions of success and failure can affect perceptions and policies relative to the status and advancement of the field of tissue engineering.

Epidermal caspase-3 cleavage associated with interferon-gamma-expressing lymphocytes in acute atopic dermatitis lesions

Keratinocyte apoptosis mediated by Fas/Fas ligand molecular interactions and subsequent caspase activation is believed to play an important role in the pathogenesis of atopic dermatitis (AD), in particular for the formation of spongiosis. To estimate epidermal caspase activation in normal and AD skin under in vivo conditions, we analysed caspase-3 cleavage by immunohistology. In normal skin as well as non-lesional AD skin, we detected caspase-3 cleavage in single cells of the basal layer. In contrast, in acute lesional AD skin, we not only obtained evidence for increased expression of cleaved caspase-3 in keratinocytes of the basal layer but also observed caspase-3 cleavage in one or more layers of the spinous cell layer, in particular in spongiotic areas. Short-term topical treatment of the skin lesions with tacrolimus or pimecrolimus abolished the expression of cleaved caspase-3 in the spinous layer. Moreover, epidermal caspase-3 cleavage correlated with the numbers of dermal interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ lymphocytes in skin lesions of AD patients, supporting the view that IFN-gamma is important for the activation of proapoptotic pathways in keratinocytes. This is also confirmed by the observation of increased Fas expression on keratinocytes in acute AD lesions that was markedly reduced following topical calcineurin inhibitor treatment. These data suggest that caspase-3 cleavage in the spinous layer of the epidermis is a pathologic event contributing to spongiosis formation in AD, whereas cleavage of caspase-3 in basal cells might represent a physiologic mechanism within the process of epidermal renewal.

Diastolic dysfunction in human cardiac allografts is related with reduced SERCA2a gene expression

Previous studies demonstrated that impaired left ventricular (LV) relaxation in cardiac allografts limits exercise tolerance post-transplant despite preserved systolic ejection fraction (EF). This study tested in human cardiac allografts whether the isovolumic relaxation time (IVRT), which provides the basis for most of diastolic LV filling, relates with gene expression of regulatory proteins of calcium homeostasis or cardiac matrix proteins. Gene expression was studied in 31 heart transplant recipients (25 male, 6 female) 13-83 months post-transplant with LVEF >50%, LV end-diastolic pressure <20 mmHg, normal LV mass index and without allograft rejection or significant cardiac pathology. IVRT related with the other diastolic parameters e-wave velocity (r = -0.46; p = 0.01), e/a-wave ratio (r = -0.5; p < 0.01) but not with heart frequency (r = -0.16; p = 0.4). No relation of IVRT was observed for immunosuppression, mean rejection grade or other medication. IVRT was not related with gene expression of desmin, collagen I, phospholamban, the Na+-Ca2+ exchanger, the ryanodine receptor or interstitial fibrosis but correlated inversely with SERCA2a (r = -0.48; p = 0.02). Prolonged IVRT is associated with decreased SERCA2a expression in cardiac allografts without significant other pathology. Similar observations in non-transplanted patients with diastolic failure suggest that decreased SERCA2a expression is an important common pathomechanism.