Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross-talk by intravital microscopy, large-scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.     

Morphometric analysis of the uvula in patients with sleep-related breathing disorders

The upper-airway mucosa in obstructive sleep apnea (OSA) patients and snorers is often described as edematous and hyperplastic. The morphologic aspects of the pharyngeal mucosa, and in particular the mucosa of the uvula and soft palate, in OSA patients are, however, not well described. The aim of the present retrospective study therefore was to perform histologic examination of the pharyngeal mucosa obtained from patients with various forms of sleep-related breathing disorders, including primary snoring. A midsagittal section of uvulas obtained by uvulopalatopharyngoplasty (UPPP) was investigated in 34 OSA patients and 9 non-apneic snorers. Control tissues were taken by autopsy in 19 patients not known to have OSA or snoring. A morphometric point counting technique was used to determine the tissue composition. The data showed that OSA patients and non-apneic snorers had a significantly greater percentage of intercellular space than controls (65.7% vs 54.0%; P = 0.006). Control uvulas contained more muscle than OSA and snorers (14.0% vs 7.8%; P = 0.006). Moreover, the covering epithelium was significantly thicker in OSA and snorers than in controls (variance ratio = 7.64; P = 0.008). When taking body mass index (BMI) into account, no correlation was found between fat deposition and BMI. Findings showed that an increased clinical severity of OSA did not affect the tissue composition. Indeed, uvula morphology was similar in OSA patients with respect to non-apneic snorers. Since the increased amount of intercellular space is the expression of edema, we hypothesize that these mucosal changes together with hyperplasia of the covering epithelium are secondary effects to snoring. They presumably play a minor role in the etiopathogenesis of OSA, but may increase the severity of OSA by further narrowing the pharyngeal lumen. Received: 18 May 1999 / Accepted: 2 July 1999     

Von der Äthernarkose zur „grünen“ Anästhesie

Die Anaesthesiologie - Vor 175 Jahren fand die erste öffentliche Demonstration einer Äthernarkose statt. Seit diesem Zeitpunkt ist Schmerzunempfindlichkeit bei chirurgischen...     

T4 lung tumors with infiltration of the thoracic aorta: is an operation reasonable?

BACKGROUND: Only anecdotal reports about the results of combined resection of T4 lung tumors infiltrating the thoracic aorta exist. METHODS: Seven patients (mean age, 57.5 years; range, 43 to 78 years) underwent a resection of the infiltrated segment of the thoracic aorta together with a left pneumonectomy (n = 6) or left upper lobectomy (n = 1). Five tumors were primary non-small cell lung carcinomas (T4N2 in 3 patients, T4N1 in 2), one was a metastasis of breast cancer, and one was rhabdomyosarcoma. RESULTS: No patient died perioperatively. The 2 patients with rhabdomyosarcoma and metastasis of breast cancer died 2 and 7 months postoperatively. Of the 5 patients with bronchial carcinoma, 3 died after 17, 26, and 27 months as a result of distant metastasis. Two patients are alive after 14 and 50 months without evidence of disease recurrence. One-year, 2-year, and 4-year survival rates for patients with bronchial carcinoma were 100%, 75%, and 25%, respectively. CONCLUSIONS: Combined resection of the lung and thoracic aorta can be performed with low morbidity and mortality when offered to highly selected patients. Adequate local control of tumor can be achieved for N1 and single-level N2 non-small cell lung carcinomas, but not for tumors with other histologies.     

Renal hemodynamic effects of L-arginine and sodium nitroprusside in heart transplant recipients

BACKGROUND: Long-term treatment with cyclosporine A (CsA) induces vasoconstriction in the kidney and causes renal impairment. An altered L-arginine (L-Arg)/nitric oxide (NO) pathway may play a key role in CsA nephrotoxicity. METHODS: We studied the effect of L-Arg (dosage, 17 mg/kg/min over 30 min), the precursor of NO synthesis, and sodium nitroprusside (SNP; dosage, 1.0 microgram/kg/min over 30 min) on renal hemodynamics in a double-blind, placebo-controlled, randomized, three-way cross-over study comprising 12 stable cardiac transplant recipients on long-term CsA treatment, 10 patients with chronic nephropathy not receiving CsA, and 13 healthy controls. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were measured by paraaminohippurate (PAH) and the inulin clearance method, respectively. RESULTS: In healthy subjects, L-Arg induced an increase in RPF (P = 0.009) and GFR (P = 0.001). By contrast, L-Arg did not induce renal hemodynamic effects in heart transplant patients or patients with chronic nephropathy. SNP reduced RPF (P = 0.050) and GFR (P = 0.005) in patients with chronic nephropathy but did not affect renal hemodynamics in heart transplant recipients or in healthy subjects. CONCLUSIONS: These data indicate that L-Arg cannot be used to reverse CsA-induced renal vasoconstriction in heart transplant recipients under long-term CsA treatment, although these patients have a normal renal response to SNP.     

Detection and Typing of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks Attached to Human Skin by PCR

Live Ixodes ricinus ticks attached to humans residing in Germany were examined for borreliae by dark-field microscopy and PCR. Borrelia species were identified by 16S rRNA sequence analysis, which showed the presence of several species, some not yet defined, and a high prevalence of multiply infected ticks.     

Sequential expression of adhesion and costimulatory molecules in graft-versus-host disease target organs after murine bone marrow transplantation across minor histocompatibility antigen barriers.

Graft-versus-host disease (GVHD) is a potentially fatal complication after allogeneic bone marrow transplantation. However, few data exist thus far on the molecular signals governing leukocyte trafficking during the disease. We therefore investigated the sequential pattern of distinct adhesion, costimulatory, and apoptosis-related molecules in GVHD organs (ileum, colon, skin, and liver) after transplantation across minor histocompatibility barriers (B10.D2 --> BALB/c, both H-2d). To distinguish changes induced by the conditioning regimen from effects achieved by allogeneic cell transfer, syngeneic transplant recipients (BALB/c --> BALB/c) and irradiated nontransplanted mice were added as controls. Irradiation upregulated the expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-l, and B7-2 in ileum, as well as VCAM-1 and B7-2 in colon, on day 3 in all animals. Whereas in syngeneic mice these effects were reversed from day 9 on, allogeneic recipients showed further upregulation of VCAM-1, ICAM-1, B7-1, and B7-2 in these organs on day 22, when GVHD became clinically evident. Infiltration of CD4+ and CD8+ donor T cells was noted on day 9 in skin and liver and on day 22 in ileum and colon. Surprisingly, the expression of several other adhesion molecules, such as ICAM-2, platelet-endothelial cell adhesion molecule 1, E-selectin, and mucosal addressin cell adhesion molecule 1, did not change. Proapoptotic and antiapoptotic markers were balanced in GVHD organs with the exception of spleen, in which a preferential expression of the proapoptotic Bax could be noted. Our results indicate that irradiation-induced upregulation of VCAM-1, ICAM-1, and B7-2 provides early costimulatory signals to incoming donor T cells in the intestine, followed by a cascade of proinflammatory signals in other organs once the alloresponse is established.     

Histochemical localization of complex carbohydrates in the nasolabial glands of the Japanese deer (Cervus nippon yakushimae)

The localization of complex glycoconjugates in the nasolabial glands of the Japanese deer (C. nippon yakushimae) was studied using various histochemical methods, including lectin histochemistry, viewed using both light and electron microscopy. The secretory epithelium and luminal secretion of the deer nasolabial glands exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-D-mannose, alpha-N-acetyl-D-galactosamine, beta-D-galactose, beta-N-acetyl-D-glucosamine and sialic acid). Additionally, O-acetylated sialic acids were detectable in the glandular acini. The results obtained are discussed with regard to the specific functions of the glandular secretion, which may particularly improve water retention on the skin surface and protect against physical damage as well as microbial contamination. Furthermore, our results support the view of a salivary nature of this gland type.     

Association between polymorphisms in caspase recruitment domain containing protein 15 and allergy in two German populations

BACKGROUND: Early exposure to microbial matter such as LPS may influence the development of asthma and allergies by activation of innate immunity pathways as indicated by studies in farming environments. Recently, polymorphisms in caspase recruitment domain containing protein 15 (CARD15), an intracellular LPS receptor protein, have been associated with Crohn's disease. Because these polymorphisms lead to changes in LPS recognition, they may affect the development of asthma and allergies. OBJECTIVE: We genotyped a large population of German schoolchildren (N = 1872) from East and West Germany for 3 functional relevant CARD15 polymorphisms for their role in the development of asthma and allergy. METHODS: By use of parental questionnaires, skin prick testing, pulmonary function tests, bronchial challenge tests, and measurements of serum IgE levels, children were phenotyped for the presence of atopic diseases. Genotyping was performed with PCR-based restriction enzyme assays. To assess associations between atopic phenotypes and genotypes standard statistical procedures were applied. RESULTS: Children with the polymorphic allele C2722 had a more than 3-fold risk to develop allergic rhinitis (P <.001) and an almost 2-fold risk for atopic dermatitis (P <.05). Furthermore, the T2104 allele was associated with an almost 2-fold risk for allergic rhinitis (P <.05). When a C insertion at position 3020 was present, the risk of atopy increased by 50% (P <.05) and serum IgE levels were elevated (P <.01). CONCLUSION: The shared genetic background between Crohn's disease and atopy may indicate that an impaired recognition of microbial exposures results in an insufficient downregulation of excessive immune responses, giving rise to either T(H)2 dominated allergies or T(H)1 related Crohn's disease.