Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

Basophils promote innate lymphoid cell responses in inflamed skin.

Type 2 inflammation underlies allergic diseases such as atopic dermatitis, which is characterized by the accumulation of basophils and group 2 innate lymphoid cells (ILC2s) in inflamed skin lesions. Although murine studies have demonstrated that cutaneous basophil and ILC2 responses are dependent on thymic stromal lymphopoietin, whether these cell populations interact to regulate the development of cutaneous type 2 inflammation is poorly defined. In this study, we identify that basophils and ILC2s significantly accumulate in inflamed human and murine skin and form clusters not observed in control skin. We demonstrate that murine basophil responses precede ILC2 responses and that basophils are the dominant IL-4-enhanced GFP-expressing cell type in inflamed skin. Furthermore, basophils and IL-4 were necessary for the optimal accumulation of ILC2s and induction of atopic dermatitis-like disease. We show that ILC2s express IL-4Rα and proliferate in an IL-4-dependent manner. Additionally, basophil-derived IL-4 was required for cutaneous ILC2 responses in vivo and directly regulated ILC2 proliferation ex vivo. Collectively, these data reveal a previously unrecognized role for basophil-derived IL-4 in promoting ILC2 responses during cutaneous inflammation.

Exposure to food allergens through inflamed skin promotes intestinal food allergy through the thymic stromal lymphopoietin-basophil axis.

BACKGROUND Exposure to food allergens through a disrupted skin barrier has been recognized as a potential factor in the increasing prevalence of food allergy. OBJECTIVE We sought to test the immunologic mechanisms by which epicutaneous sensitization to food allergens predisposes to intestinal food allergy. METHODS Mice were epicutaneously sensitized with ovalbumin or peanut on an atopic dermatitis-like skin lesion, followed by intragastric antigen challenge. Antigen-specific serum IgE levels and T(H)2 cytokine responses were measured by ELISA. Expression of type 2 cytokines and mast cell proteases in the intestine were measured by using real-time PCR. Accumulation of basophils in the skin and mast cells in the intestine was examined by using flow cytometry. In vivo basophil depletion was achieved by using diphtheria toxin treatment of Baso-DTR mice. For cell-transfer studies, the basophil population was expanded in vivo by means of hydrodynamic tail vein injection of thymic stromal lymphopoietin (TSLP) cDNA plasmid. RESULTS Sensitization to food allergens through an atopic dermatitis-like skin lesion is associated with an expansion of TSLP-elicited basophils in the skin that promote antigen-specific T(H)2 cytokine responses, increased antigen-specific serum IgE levels, and accumulation of mast cells in the intestine, promoting the development of intestinal food allergy. Critically, disruption of TSLP responses or depletion of basophils reduced the susceptibility to intestinal food allergy, whereas transfer of TSLP-elicited basophils into intact skin promoted disease. CONCLUSION Epicutaneous sensitization on a disrupted skin barrier is associated with accumulation of TSLP-elicited basophils, which are necessary and sufficient to promote antigen-induced intestinal food allergy.

Memory regulatory T cells reside in human skin.

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

Monogenic Human Skin Disorders

Human genodermatoses represent a broad and partly confusing spectrum of countless rare diseases with confluent and overlapping phenotypes often impeding a precise diagnosis in an affected individual. High-throughput sequencing techniques have expedited the identification of novel genes and have dramatically simplified the establishment of genetic diagnoses in such heterogeneous disorders. The precise genetic diagnosis of a skin disorder is crucial for the appropriate counselling of patients and their relatives regarding the course of the disease, prognosis and recurrence risks. Understanding the underlying pathophysiology is a prerequisite to understanding the disease and developing specific, targeted or individualized therapeutic approaches. We aimed to create a comprehensive overview of human genodermatoses and their respective genetic aetiology known to date. We hope this may represent a useful tool in guiding dermatologists towards genetic diagnoses, providing patients with individual knowledge on the respective disorder and applying novel research findings to clinical practice.

Late diagnosis of fucosidosis in a child with progressive fixed dystonia, bilateral pallidal lesions and red spots on the skin

Fucosidosis is a rare lysosomal storage disease. A 14-year-old girl is presented, with recurrent infections, progressive dystonic movement disorder and mental retardation with onset in early childhood. The clinical picture was also marked by mild morphologic features, but absent dysostosis multiplex and organomegaly. MRI images at 6.5 years of age were reminiscent of pallidal iron deposition ("eye-of-the-tiger" sign) seen in neurodegeneration with brain iron accumulation (NBIA) disorders. Progressively spreading angiokeratoma corporis diffusum led to the correct diagnosis. This case extends the scope of clinical and neuroradiological manifestations of fucosidosis.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs.

Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism Anaplasma phagocytophilum. CGA is typically characterized by fever, thrombocytopenia, lethargy, anorexia, arthropy, and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans. The pathophysiology of CGA is not entirely clear, and the persistence of the organism after the resolution of clinical signs has been described. The aim of the study was to investigate if A. phagocytophilum can be detected in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline. Paraffin-embedded lesional skin biopsies were allocated into separate groups: biopsies from A. phagocytophilum-seropositive dogs responsive to treatment with doxycycline (n=12), biopsies from A. phagocytophilum-seronegative dogs (n=2), and biopsies in which skin lesions histopathologically resembled a tick bite (n=10). The serological status of the latter group was unknown. Histology of the seropositive and seronegative dog skin lesions did not indicate an etiology. DNA was extracted, and a conventional PCR for partial 16S rRNA gene was performed. Anaplasma phagocytophilum DNA was amplified from 4/12 seropositive dogs' skin biopsies. All sequences were 100% identical to the prototype A. phagocytophilum human strain (GenBank accession number U02521). Anaplasma phagocytophilum was not amplified from the 2 seronegative and 10 suspected tick bite dogs. Serum antibody titers of the PCR-positive dogs ranged from 1:200 to 1:2048. Histopathologically, a mild-to-moderate perivascular to interstitial dermatitis composed of a mixed cellular infiltrate and mild-to-moderate edema was seen in all seropositive dogs. In 8/12 seropositive dogs, vascular changes as vasculopathy, fibrinoid necrosis of the vessel walls, and leukocytoclastic changes were observed. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs.

The immunology of the porcine skin and its value as a model for human skin.

The porcine skin has striking similarities to the human skin in terms of general structure, thickness, hair follicle content, pigmentation, collagen and lipid composition. This has been the basis for numerous studies using the pig as a model for wound healing, transdermal delivery, dermal toxicology, radiation and UVB effects. Considering that the skin also represents an immune organ of utmost importance for health, immune cells present in the skin of the pig will be reviewed. The focus of this review is on dendritic cells, which play a central role in the skin immune system as they serve as sentinels in the skin, which offers a large surface area exposed to the environment. Based on a literature review and original data we propose a classification of porcine dendritic cell subsets in the skin corresponding to the subsets described in the human skin. The equivalent of the human CD141(+) DC subset is CD1a(-)CD4(-)CD172a(-)CADM1(high), that of the CD1c(+) subset is CD1a(+)CD4(-)CD172a(+)CADM1(+/low), and porcine plasmacytoid dendritic cells are CD1a(-)CD4(+)CD172a(+)CADM1(-). CD209 and CD14 could represent markers of inflammatory monocyte-derived cells, either dendritic cells or macrophages. Future studies for example using transriptomic analysis of sorted populations are required to confirm the identity of these cells.

Lung Nodule Detection by Microdose CT Versus Chest Radiography (Standard and Dual-Energy Subtracted).

OBJECTIVE The purpose of this study was to investigate the feasibility of microdose CT using a comparable dose as for conventional chest radiographs in two planes including dual-energy subtraction for lung nodule assessment. MATERIALS AND METHODS We investigated 65 chest phantoms with 141 lung nodules, using an anthropomorphic chest phantom with artificial lung nodules. Microdose CT parameters were 80 kV and 6 mAs, with pitch of 2.2. Iterative reconstruction algorithms and an integrated circuit detector system (Stellar, Siemens Healthcare) were applied for maximum dose reduction. Maximum intensity projections (MIPs) were reconstructed. Chest radiographs were acquired in two projections with bone suppression. Four blinded radiologists interpreted the images in random order. RESULTS A soft-tissue CT kernel (I30f) delivered better sensitivities in a pilot study than a hard kernel (I70f), with respective mean (SD) sensitivities of 91.1% ± 2.2% versus 85.6% ± 5.6% (p = 0.041). Nodule size was measured accurately for all kernels. Mean clustered nodule sensitivity with chest radiography was 45.7% ± 8.1% (with bone suppression, 46.1% ± 8%; p = 0.94); for microdose CT, nodule sensitivity was 83.6% ± 9% without MIP (with additional MIP, 92.5% ± 6%; p < 10(-3)). Individual sensitivities of microdose CT for readers 1, 2, 3, and 4 were 84.3%, 90.7%, 68.6%, and 45.0%, respectively. Sensitivities with chest radiography for readers 1, 2, 3, and 4 were 42.9%, 58.6%, 36.4%, and 90.7%, respectively. In the per-phantom analysis, respective sensitivities of microdose CT versus chest radiography were 96.2% and 75% (p < 10(-6)). The effective dose for chest radiography including dual-energy subtraction was 0.242 mSv; for microdose CT, the applied dose was 0.1323 mSv. CONCLUSION Microdose CT is better than the combination of chest radiography and dual-energy subtraction for the detection of solid nodules between 5 and 12 mm at a lower dose level of 0.13 mSv. Soft-tissue kernels allow better sensitivities. These preliminary results indicate that microdose CT has the potential to replace conventional chest radiography for lung nodule detection.

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Quantitative PCR for the diagnosis of cutaneous leishmaniasis from formalin-fixed and paraffin-embedded skin sections.

The present report describes a real-time PCR-based procedure to reliably determine the quantity of Leishmania amastigotes in relation to the amount of host tissue in histological skin sections from canine and equine cases of cutaneous leishmaniasis. The novel diagnostic Leishmania-PCR has a detection limit of <0.02 amastigotes per μg tissue, which corresponds well to the detection limit of immunohistochemistry and is far beyond that of conventional histology. Our results emphasise the importance of PCR to complement routine histology of cutaneous leishmaniasis cases, particularly in laboratories in which no immunohistochemical assay is available.