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The transient receptor potential canonical 6 (TRPC6) channel has been studied in pathologies of the hepatobiliary system. Information on the localization of TRPC6-protein in anatomical and histological structures of the human hepatobiliary system in elderly with and without liver disease is lacking.Samples were obtained from twelve nitrite pickling salt-ethanol-polyethylene glycol-fixed corpses of the four anatomical liver lobes, collum and the remaining gallbladder, the common bile duct (gender: 5 females, 41.67%; age [years]: median 84, range 20; postmortem interval before fixation [h]: median 60, range 74.15). Orienting histomorphologic assessment was done in a hematoxylin-eosin stain. Immunohistochemical labelling of TRPC6 was carried out following a cross-evaluation scheme. TRPC6-immunoreactivity was assessed regarding intensity (semi-quantification, three independent observers) and area (optical threshold method). Explorative statistical analyses were performed using R (inter-rater-reliability: Fleiss' kappa; comparisons: Wilcoxon-Rank-sum-test).Extensive autolysis was found in the liver of one and the biliary samples of three corpses. Extensive liver disease was found in 4 corpses, while 8 corpses only showed age-appropriate degeneration. Only the intrahepatic connective tissue showed no TRPC6-immunoreactivity. Individuals with extensively diseased livers exhibited statistically significantly less TRPC6-immunoreactive area in the bile duct and liver tissue while statistically significant more TRPC6-immunoreactive area in the gallbladder compared to individuals with age-appropriate degeneration of the liver only in the respective organ.Age-associated degeneration of the hepatobiliary system is likely to be associated with widespread, homogenous TRPC6-expression. Liver disease potentially influences the distribution of TRPC6-protein within the liver and within the biliary tract in elderly.

Introduction Pending changes regarding the medical curricula in Germany prompted us, members of the Department of Anatomy at the Medical Faculty of Saarland University, to critically evaluate anatomy teaching with special attention to macroscopic anatomy and the dissection class. Aiming at a thorough assessment we were particularly interested to which extend an optional peer-led tutorial (“anatomy boot camp”), which had preceded the dissection class, impacted the outcome in a series of oral exams of those who had participated. Methods We evaluated a student cohort of 307 students in the fall/winter semester 2015/16 at Saarland University, including those enrolled in medicine and dentistry, by implementing a series of questionnaires specifically designed for five mandatory oral exams during the course of the dissection class. The questionnaires contained different exam-relevant questions as well as several demographic items. The evaluation was complemented by interviews with students and examiners. Results Participants of the anatomy boot camp generally performed better in all exams and in three of them statistically significant. Items such as gender, age, and previous experiences in the health care professions contributed marginally – if at all – to the overall students’ performance. Intriguingly, most students welcomed the idea of implementing questionnaire-based oral exams to balance out the different experience level of the respective examiner. Conclusion Our study showed that a comparable minimal timely investment on the part of the students to choose to participate in an optional anatomy boot camp was positively correlated with students’ performance.

Due to longer lifespans in societies in industrialized countries, cardiovascular diseases are becoming increasingly important for medical research. It has already been shown that the cell membrane-bound, non-selective TRPC6 ion channel is important in the pathogenesis of heart diseases. Among other things, it is permeable to calcium ion, which plays a critical role in cardiac contraction and relaxation. The TRPC6 ion channel is a promising therapeutic target in the treatment of cardiovascular diseases. A deeper understanding of the physiological and pathophysiological role as well as the localization of TRPC6 in human cardiac tissue is the basis for new drug development. Although the TRPC6 channel has been detected in animal studies, at the mRNA level in humans, and sparse TRPC6 protein has been detected in humans, there are no systematic studies of TRPC6 protein detection in the human heart. For the first time, TRPC6 ion channel protein was detected histologically in human heart tissue from body donors in different structures, localizations, and histological layers - particularly in cardiomyocytes and intramuscular arterioles - by immunohistochemistry, just as TRPC6 expression has already been shown in animal models of the heart by other research groups. In the sense of the translational concept, this indicates a possible transferability of research results from animal models to humans.

Different techniques are available for carpal tunnel release such as classical surgical operations or minimally invasive procedures. A minimally invasive approach and the relevant regional anatomy are reported in this pilot study which was conducted with bodies from a body donation program. The method described here uses a hook knife to cut through the transverse carpal ligament or flexor retinaculum under ultrasound guidance. The results are documented by means of magnetic resonance and ultrasound imaging as well as by anatomical dissection.

INTRODUCTION The purpose of this study was to evaluate the effect of posterior septectomy size on surgical exposure and surgical freedom during the endoscopic transsphenoidal approach to the sellar and parasellar region. METHODS A mononostril and binostril approach to the sellar region was performed on 4 formalin-fixed cadaveric heads. Predefined anatomical structures were identified. Additionally, a millimeter gauge was introduced into the surgical site and the extent of dorsal septectomy was analyzed for both approaches. Surgical freedom was defined as the distance between the ipsilateral and contralateral limit of opening of the sphenoid sinus. RESULTS The mean extent of dorsal septectomy was 15.7±5.7mm using a binostril approach to achieve adequate visualization of all relevant anatomical structures. Superior results were obtained via binostril technique with respect to the ability to identify the contralateral internal carotid artery or opticocarotid recessus. No such advantage was found for all other landmarks. Surgical freedom between the ipsilateral and contralateral limit of exposure of the sphenoid sinus was measured with 15±0.8mm in the mononostril and 19.2±0.9mm in the binostril group. CONCLUSIONS The surgical exposure increased significantly with progressively larger posterior septectomy in binostril approaches until a 20-mm posterior septectomy. Bilateral lateral opticocarotid recesses were accessible with a mean of 15mm for posterior septectomy. In the mononostril group no dorsal septectomy was necessary. Thus, the nasal mucosa is more preserved by this technique. However, the lateral exposure is partially limited and the use of angled endoscopes is recommended when adopting a mononostril approach to the sellar region.

Background Knowledge of the anatomical dimensions of distinct areas of the vertebral bodies and vertebral arches of the subaxial cervical spine are indispensable in the planning of osteosynthesis. The minute dimensions and complex anatomical orientation of the posterior vertebral arch structures in the subaxial spine render the insertion of screws - where needed - a challenging procedure. The pedicle option carries the risk of serious complications while the alternative of lateral mass only permits the insertion of short screws. Objective The transverse process of the subaxial vertebrae offers unique possibilities on all counts and seems quite comparable in its dimensions with the pedicle. To our knowledge it has not been used previously for the insertion of screws in the subaxial spine. Methods Therefore, the scans of 66 cervical spines were analysed for distinct lines of both structures. Results The widths and lengths of the dorsal part of the transverse processes and of the pedicles are similar between both sides. Clear differences between females and males could be observed. The widths of both structures were closer to each other in C3 than in C4 to C7, while the lengths derived the most in C7. Conclusion The dorsal part of the transverse process might be suitable for the insertion of screws to stabilize the vertebral arch of the subaxial cervical spine. Gender adaptation might be required.

This is a short view point on a possible role of physician's pledges in today's medical careers. The history of the Hippocratic Oath and the Geneva Declaration was depicted and its relevance for education in Anatomy was mooted. Anatomy is taught very early in the medical curriculum and anatomists should behave in an exemplary manner with respect to the dead bodies as well as to medical students as future colleagues.

Acetylcholine is synthetized and released from neural cells, but also by non-neuronal cells such as epithelial cells or keratinocytes. Cholinergic agonists enhance the phagocytosis of zymosan particles in primary peritoneal macrophages. The aim of this study was to investigate the effect of carbachol stimulation on phagocytosis in a macrophage cell line using microspheres. The murine cell line MH-S was used in a phagocytosis assay with fluorescent latex beads. The amount of the ingested beads was determined using flow cytometry. Gene expression was investigated using polymerase chain reaction. Gene expression of the muscarinic receptors M1, M3, M4 and M5 but not M2 was found. Carbachol slightly increased the phagocytosis of microspheres in the macrophages. A co-stimulation using lipopolysaccharide and carbachol did not increase the effect of lipopolysaccharide alone. In conclusion, cholinergic stimulation in vitro only moderately modulates the phagocytosis of microspheres. M2 might have a role in stimulation of macrophage phagocytosis.

Introduction Bone decalcification is a necessary preprocessing step in histological and anatomical studies. Several solutions for decalcification with different claimed times for full decalcification are commercially available. Current literature lacks direct, quantitative measurement of calcium hydrocyapatite degradation during decalcification to compare different solutions. Therefore, the aim of this study was to test the performance of three different decalcification solutions in human bone by direct measurement of calcium hydroxyapatite using dual-X-ray-absorptiometry (DEXA) and volumetric computed tomography (CT). Methods Four femur slices were acquired from the proximal femur of a 76-year-old body donor. The slices were submerged in formaldehyde (control), EDTA, Osteosoft (Merck, Darmstadt, Germany) and “Rapid Bone Decalcifier” (RBD) (American MasterTech Scientific, Lodi, USA). Consecutive DEXA and CT scans were performed at 2 h, 4 h, 8 h, 11 h, 20 h, 44 h and 77 h after solutions were added. Besides the calcium hydroxyapatite concentration, the bone volume was measured each time. Results Fastest decline in volume was seen in the RBD probe. Further, RBD was the only solution, being able to fully decalcify the bone slice after 77 h. Although a steady decline in volume and hydroxyapatite concentration was seen for EDTA and Osteosoft as well, both were not able to decalcify the slices. Conclusion Overall, the purely qualititve acquired literature data on bone decalcifiers was verified by our quantitative data for human, cortical-rich bones. Hydrochloric-acid based solutions seem to be preferable in order to rapidly dissolve the calcium hydroxyapatite.

Background & Aims Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can progress to non-alcoholic steatohepatitis (NASH). The identification of molecular and cellular factors that determine the progression of NASH and lead to irreversible hepatocellular damage are crucial. Dendritic cells (DCs) represent a heterogeneous cell population among which CD103 + DCs play a significant role in immunity and tolerance. We aimed to clarify the role of this DC subset in the pathomechanism of NASH. Methods Steatosis progression towards steatohepatitis was analysed using multicolor FACS analyses, cytokine and qPCR array in high sucrose diet (HSD) and methionine and choline deficient diet (MCD) fed wild-type and basic leucine zipper transcription factor, ATF-Like-3 ( Batf3 ) deficient animals, which lack CD103 + DCs (classical type-1 DC, cDC1s). Results Metabolic challenge of Batf3 −/− animals resulted in the progression of steatosis towards steatohepatitis, manifesting by an increased influx of inflammatory cells into the liver and elevated inflammatory cytokine production of myeloid cells upon innate stimuli. However, the lack of cDC1s did not affect cellular apoptosis and fibrosis progression but altered genes involved in lipid metabolism. The adoptive transfer of CD103 + cDC1s to Batf3 deficient animals reversed these observed changes and more importantly could attenuate cellular damage and inflammation in established murine steatohepatitis. Conclusion Here, we have identified the murine CD103 + cDC1s as a protective DC subtype that influences the pro–anti-inflammatory balance and protects the liver from metabolic damage. As guardians of liver integrity, they play a key role in the inflammatory process during the development of steatohepatitis in mice. Lay summary Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can lead to non-alcoholic steatohepatitis (NASH). The current study demonstrated that a specific murine dendritic cell subtype possesses a potent regulatory role to influence the inflammatory milieu of the liver in this process.

Perinatal hypoxia is a critical complication during delivery and is mostly studied in animal models of postnatal hypoxic-ischemic brain injury. We here studied the effects of postnatal hypoxic-ischemic brain injury in two different sub-strains of C57BL/6 mice, i.e. C57BL/6J and C57BL/6N mice. These two sub-strains show different metabolic properties, for instance an impaired glucose tolerance in C57BL/6J mice. Genetically, this was linked to differences in their nicotinamide nucleotide transhydrogenase (Nnt) genes: In C57BL/6J mice, exons 7–11 of the Nnt gene are deleted, resulting in the absence of functional Nnt protein. The mitochondrial Nnt-protein is one of several enzymes that catalyses the generation of NADPH, which in turn is important for the elimination of reactive oxygen species (ROS). As ROS is thought to contribute to the pathophysiology of hypoxia-ischemia, the lack of Nnt might indirectly increase ROS levels and therefore result in increased brain damage. We therefore hypothesize that lesion score and lesion size will increase in C57BL/6J mice as compared to C57BL/6N mice. Surprisingly, the results showed exactly the opposite: C57BL/6J mice showed a decrease in lesion score and size, associated with a reduced number of apoptotic cells and activated microglia. In contrast, the number of cells with ROS-induced DNA modifications (detected by 8OHdG) was higher in C57BL/6J than C57BL/6N mice. In conclusion, C57BL/6J mice showed reduced ischemic consequences after postnatal hypoxic-ischemic brain injury compared to C57BL/6N mice, with the exception of the amount of ROS-induced DNA-damage. These differences might relate to the lack of Nnt, but also to a modified metabolic setting (cardiovascular parameters, oxygen and glucose metabolism, immune function) in C57BL/6J mice.

The anterior abdominal fat pad is associated with the falciform ligament in the upper middle/right abdomen and is frequently seen there in diagnostic imaging. It varies greatly in size and has often been described as an incidental finding in adults and has hitherto rarely been regarded as being illness-relevant. The aim of this study has been to assess whether the dimension of the corpus adiposum may be associated with body mass index. Ultrasound findings of 26 patients from birth until adolescence were analyzed for this purpose. In addition, an example from a recent dissection course has been included. The structure is constantly found with its smallest dimension in newborns, with a slight increase in infancy. The average dimensions were 7.6 by 3.5 by 0.7cm. The cubic volume correlated with age, weight and body mass index, whereas the latter association was strongest. Our data suggest that routinely determined dimension of falciform fat may be a surrogate parameter of relative body weight in childhood.

MALP-2, a synthetic lipopeptide is a Toll-like receptor 2 and -6 ligand and agonist. MALP-2 stimulates immune cells at different sites. Local stimulation in the lungs has beneficial effects in experimental pneumococci infection. The presented study investigated local effects of MALP-2 in the mycobacterial infection of lungs. MALP-2 was applied prior, simultaneously or after the pulmonary infection with Mycobacterium bovis BCG. Colony forming units were determined in the bronchoalveolar lavage fluid and lung homogenate. Numbers of Mycobacterium bovis BCG colony forming units were found to be reduced in two compartments, bronchoalveolar lavage fluid and lung homogenate after treatment with MALP-2 simultaneously to the infection for up to eight weeks. Reduction of the bacterial load in both compartments was also found up to two weeks after local treatment before and after the infection. Thus, macrophage activating lipopeptide-2 enhances the host defence in the lung in acute and long term bacterial infections.

Nicotinamide-nucleotide-transhydrogenase (Nnt) is a mitochondrial protein. It is altered and functionally lacking in the C57BL/6J sub-strain. This leads to the generation of more radical oxygen species than in the C57BL/6N sub-strain. During studies on the effect of Nnt in perinatal hypoxia the cerebral vasculature was investigated in postnatal day 9 mice using post mortem arterial filling with silicone rubber compounds. Surprisingly, the tiny vessels were no longer uniformly filled and a bleb-like pattern occurred in both sub-strains. Furthermore, considerably more bleb-like spots were observed in the C57Bl/6J sub-strain than in the C57Bl/6N sub-strain. These blebs might be the result of feathery vessels bursting. It remains unclear how the mechanisms in the used strains differ. Nnt might influence the vascular structure or its development and mechanisms and should be investigated further.

In this Short Communication, the issue is discussed whether the recently reported anterolateral ligament of the knee is a unique structure or just part of the iliotibial tract. The presented findings from investigations in body donors support the view of a unique structure.

Janus kinase (JAK) inhibitors have recently been developed for allergic diseases. We focused on the 2 different JAK inhibitors, tofacitinib (selective for JAK3) and oclacitinib (selective for JAK1 and 2), to clarify the mechanism of anti-inflammatory and anti-itching potency of these drugs. In the process of detecting anti-itching potency, we observed that tofacitinib treated mice showed aggression behaviour. The objective of the study reported here was to investigate the aggressive behaviour induced by tofacitinib by using a mouse model of allergic dermatitis and the resident-intruder test. For the allergic dermatitis model, female BALB/c mice were sensitised and challenged topically with toluene-2,4-diisocyanate (TDI). Vehicle, tofacitinib or oclacitinib, was administered orally 30 min before TDI challenge. Scratching, aggression and standing behaviours were monitored in the 60 min period immediately following challenge of TDI. Another group of male BALB/c mice treated with vehicle, tofacitinib or oclacitinib was evaluated in the resident-intruder test and brains were obtained to determine blood brain barrier penetration. In the allergic dermatitis model, a significant increase in aggression and standing behaviour was only obvious in the tofacitinib treatment group. There was no effect in non-sensitised mice, but similar aggression was also induced by tofacitinib in male resident-intruder test. Penetration of blood-brain barrier was observed both in tofacitinib and oclacitinib treated mice. These results suggest that aggression was induced by tofacitinib under some kind of stressful environment. This study indicates a possible role of the JAK-STAT pathway in modulation of aggression behaviour.

The activation of inflammasome signaling mediates pathology of acute Pseudomonas aeruginosa pneumonia. This suggests that the inflammasome might represent a target to limit the pathological consequences of acute P. aeruginosa lung infection. Pannexin-1 (Px1) channels mediate the activation of caspase-1 and release of IL-1β induced by P2X7 receptor activation. The approved drug probenecid is an inhibitor of Px1 and ATP release. In this study, we demonstrate that probenecid reduces infection and inflammation in acute P. aeruginosa pneumonia. Treatment of mice prior to infection with P. aeruginosa resulted in an enhanced clearance of P. aeruginosa and reduced levels of inflammatory mediators, such as IL-1β. In addition, probenecid inhibited the release of inflammatory mediators in murine alveolar macrophages and human U937 cell-derived macrophages upon bacterial infection but not in human bronchial epithelial cells. Thus, Px1 blockade via probenecid treatment may be a therapeutic option in P. aeruginosa pneumonia by improving bacterial clearance and reducing negative consequences of inflammation.

Summary Little is known about the normal immune cell profile in the lungs of infants without pulmonary disease. Normal lung samples obtained at autopsy of 10 infants that died either due to incidental or inflicted causes or non-pulmonary diseases were stained for antibodies against B and T lymphocytes, macrophages, NK cells, cytotoxic cells, dendritic cells and mast cells. Cells were quantified in the airway epithelial layer, inner layer (between the epithelium and the outer smooth muscle border), outer layer (between the outer smooth muscle border and the external limits of the airway) and alveolar septa. Basement membrane or alveolar septa lengths were assessed by image analysis. Results were expressed as cells/mm. The median age of patients was 6.8 months, ranging from 11 to 840 days. The inner layer of the airways was the region with the smallest density of cells. There was a predominance of cells related to the innate immunity such as CD56+, Granzyme B+ and CD68+ cells in the epithelial layer and alveolar parenchyma. The outer layer and the lung parenchyma presented the highest cellular density. There were very few CD4+ T cells or dendritic cells in most of the lung compartments. The numbers of CD3+ T and granzyme B+ cells correlated positively with age. There was a compartmentalization of immune cells along airways and parenchyma, which may be related to the development of innate and acquired lung defense mechanisms.

Nanotechnology is a rapidly developing field in science and industry. The exposure to nanoparticles (NPs) will steadily grow in the future and there is thus an urgent need to study potential impacts of the interaction between NPs and the human body. The respiratory tract is the route of entry for all accidentally inhaled NPs. Moreover, NPs may intentionally be delivered into the lung as contrast agents and drug delivery systems. The present review provides an overview of currently used techniques for the in vivo imaging of NPs in the lung, including x-ray imaging, computed tomography, gamma camera imaging, positron emission tomography, magnetic resonance imaging, near-infrared imaging, and intravital fluorescence microscopy. Studies based on these techniques may contribute to the development of novel NP-based drug delivery systems and contrast agents. In addition, they may provide completely new insights into nanotoxicological processes. From the Clinical Editor Nanoparticles are rapidly gaining ground in various therapeutic and diagnostic applications. This review provides an overview of current in vivo imaging techniques of NPs in the lung, including x-ray, CT, gamma camera imaging, PET, MRI, near-infrared imaging, and intravital fluorescence microscopy, aiding the development of novel NP-based techniques and nanotoxicology.

Migration of Langerhans cells (LCs) from the skin to the lymph node is an essential step in the pathogenesis of allergic contact dermatitis (ACD). Therefore, inhibition of LC-migration could be a promising strategy to improve this skin disease. Effects of sphingosine-1-phosphate (S1P) and the immunomodulator FTY720 on LC trafficking is not well defined, yet. Thus, we investigated the action of topically administered S1P and FTY720 in a murine model of ACD. Most interestingly, FTY720 as well as S1P inhibited the inflammatory reaction in the elicitation phase of ACD. In the sensitization phase, FTY720, and S1P reduced the weight and cell count of the draining auricular lymph node, as well as immigrated dendritic cells provoked by repetitive topical administration of the hapten. Correspondingly, the density of LCs in the epidermis was higher in FTY720- and S1P-treated mice compared to vehicle treatment. A skin dendritic cell migration assay confirmed the significant inhibition of dendritic cell migration by FTY720 and S1P. These data supply conclusive evidence that the strategy of targeting the migratory response of LCs with locally acting S1P or FTY720 represents an emerging option in the treatment of allergic skin diseases like contact hypersensitivity and atopic dermatitis.

Summary A tissue-engineered articular condyle could provide a new alternative approach to joint replacement. This study describes progress made towards engineering an articular condyle in vitro using human bone marrow stromal cells (hBMSCs) in a biphasic matrix. hBMSCs were transferred to a rat collagen-I hydrogel which was then pressed onto a bovine cancellous bone matrix. The gel/cell suspensions, each at a density of approximately 5×10 5 cells/ml containing fourth passage cells pressed into an adult human tibial condyle form using CT scan based moulds. The osteochondral constructs fabricated in vitro were stimulated in a bioreactor using cyclic compression and continuous perfusion. Penetration and cell distribution were demonstrated as homogeneous and cells were found to be viable after gel compression. The filamentous structure of the collagen fibres was more dense and homogeneous using compression. Mechanical tests showed a significant enhancement of primary matrix stability after initial compression. Stiffness was not observed to increase significantly over 7 days under loading in a bioreactor. The successful integration of mechanical stimulation in the tissue engineering process leads to an improvement in the structural and biomechanical properties of these tissues and offers new possibilities in the management of joint injuries and degenerative diseases. Remarkably, the stiffness was enhanced in our setting after initial compression of the construct in the glass cylinder without observing a negative influence on cell viability. Further studies need to clarify the influence of compression and various mechanical and hydrostatic stress patterns over different periods of time.

Acetylcholine (ACh), derived both from nerve fibres and from non-neuronal sources such as epithelial cells, is a major regulator of airway function. There is evidence that dysfunction of the neuronal cholinergic system is involved in the pathogenesis of asthma. Here, we asked whether the pulmonary non-neuronal ACh-synthesis and release machinery is altered in a rat and a mouse model of allergic airway disease. Animals were sensitized against ovalbumin, challenged by allergen inhalation, and sacrificed 24 or 48 h later. Targets of investigation were the high-affinity choline transporter-1 (CHT1), that mediates cellular uptake of choline, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the polyspecific organic cation transporters (OCT1-3), which are able to translocate choline and ACh across the plasma membrane. With cell-type specific distribution patterns, immunohistochemistry identified these proteins in airway epithelial cells and alveolar macrophages. Real-time RT-PCR revealed significant decreases in ChAT-, CHT1-, VAChT-, OCT-mRNA in the lung of sensitized and allergen challenged animals. These data were supported by immunohistochemistry, demonstrating reduced labeling intensity of airway epithelial cells. ChAT-, CHT1-, VAChT-, and OCT1-mRNA were also significantly reduced in cells recovered by bronchoalveolar lavage from sensitized and challenged rats. In conclusion, the pulmonary non-neuronal cholinergic system is down-regulated in acute allergic airway inflammation. In view of the role of ACh in maintenance of cell-cell-contacts, stimulation of fluid-secretion and of ciliary beat frequency, this down-regulation may contribute to epithelial shedding and ciliated cell dysfunction that occur in this pathological condition.

To assess published evidence of anaphylatoxin receptor expression in renal tubular epithelial cells, monoclonal antibodies (mAbs) against human, mouse and rat receptors for C5a and C3a (C5aR, C3aR) were raised using receptor-expressing transfectants as immunogens. Applying these reagents in immunohistochemistry, we observed that mAbs with reactivities against three distinct epitopes of human C5aR N-terminus recognized tissue macrophages but not at all renal tubular epithelial cells. These findings were surprising, as strong tubular staining had been previously demonstrated by mAbs raised against a synthetic N-terminal C5aR peptide. To extend our study to mammalian kidneys, renal specimens from normal rats as well as LPS-treated Balb/c and MRL/lpr mice, which suffered from lupus-type nephritis, were examined. Similar to humans, mAbs against murine or rat C5aR strongly recognized infiltrating leukocytes in situ whereas tubular epithelial cells remained negative. As a mAb has been previously used to document C3aR expression in renal tubular epithelial cells, kidney specimens were examined using newly established mAbs against different epitopes of human, murine and rat C3aR. In contrast to published evidence, C3aR was detectable exclusively in interstitial leukocytes but not in epithelial tubular cells of normal and diseased tissues. Taken together, our findings question a direct involvement of tubular epithelial cells in anaphylatoxin-mediated renal inflammation. Furthermore, as we demonstrate in the case of anaphylatoxin receptors, cross-reactivities of mAbs may constitute as yet underestimated pitfalls in immunohistochemical antigen detection.

The macrophage-activating lipopeptide with a molecular weight of 2kDa (MALP-2) activates antigen presenting cells of human, mouse and rat origin in vitro and in vivo. Here, we demonstrate that MALP-2 induces MIP1alpha and beta, MIP-2, Gro, TNFalpha, IL1alpha and IL6 in cells of cotton rats (Sigmodon hispidus) in vitro. Intranasal inoculation into cotton rats leads to migration of neutrophils and other leucocytes into the lung lumen and lung tissue. After intranasal co-inoculation of MALP-2 with live-attenuated measles vaccine virus, higher titers of neutralizing antibodies are induced but the proliferative T cell response did not increase. Immunization leads to protective immunity in the absence, but not in the presence of passively transferred measles virus (MV) specific antibodies.

Background The mechanisms leading to death from asthma are not completely understood. Recent studies suggest the involvement of the anaphylatoxins C3a and C5a, generated during complement activation, and their receptors C3aR and C5aR in the pathogenesis of asthma. Objective The aim of our study was to investigate the expression of C3aR and C5aR in fatal asthma. Methods We analyzed lung tissue from 14 subjects who died of asthma (fatal asthma; FA) and 14 subjects who died of nonpulmonary causes (controls) and bronchial biopsy specimens from 16 subjects with mild intermittent asthma (MIA). C3aR and C5aR expression was evaluated by immunohistochemistry, and a semiquantitative analysis of the intensity of staining was performed according to a visual analogue scale (score, 0-3). Results C3aR was expressed on airway epithelium, smooth muscle, submucosal, and parenchymal vessels. C5aR was expressed on myeloid cells infiltrating the submucosa and on airway epithelium. Statistical analysis demonstrated higher expression of C3aR on submucosal vessels in FA compared with controls and MIA (median [minimum-maximum], controls, 0.24 [0-1.48]; MIA, 0.0 [0-1.00]; FA, 1.56 [0.13-3]; P =.002). C3aR was also increased on parenchymal vessels in FA (controls, 0.56 [0-2.00]; FA, 1.81 [0.5-3]; P =.0004). C5aR expression on airway epithelium was increased in FA compared with controls and MIA (controls, 1.25 [0.25-3]; MIA, 1.00 [0-2.00]; FA, 3.00 [1.13-3.00]; P =.001). Conclusion The results of our study suggest a role of complement in FA.

Murine listeriosis is a paradigm to understand host pathogen interactions. Airway infections with Listeria monocytogenes, although representing a serious problem in early onset neonatal listeriosis, has not been investigated in detail in animal models so far. Here, the susceptibility of BALB/c, DBA/2 and C57BL/6 mice towards an intratracheal (i.t.) infection with virulent L. monocytogenes EGDe and the attenuated variant L. monocytogenes EGD hlyW491A(pERL3-CMVGFP) is reported. The course of infection was characterized by determination of bacterial numbers in the organs and assessment of the health condition of the mice. The distribution and cellular localization of Listeria in the airways was assessed by immunocytochemistry and confocal and electron microscopy. The differential susceptibility of inbred mouse strains to airway infections with L. monocytogenes could be assigned to the major virulence factor listeriolysin O. Resistant C57BL/6 mice were not affected by the two listerial strains. In contrast, BALB/c and DBA/2 mice showed differential susceptibility towards L. monocytogenes EGDe and attenuated bacteria, with all the mice being killed by the wild-type bacteria but rarely by the variant that secretes a listeriolysin of only 10% activity of that of the wild-type toxin. Thus, listeriolysin is a decisive factor for differential susceptibility against Listeria. After i.t. application, bacteria were predominantly localized in the peribronchiolar space and invaded alveolar macrophages but rarely lung epithelial cells. Dissemination from the lung into the deep organs started almost immediately after application, although a pulmonary bacterial reservoir remained during the first 4 days.

In asthma surfactant proteins (SP) might differ in distribution and composition and thus play a role in pathophysiology of this disease. Therefore, the well-established animal model of ovalbumin sensitized and challenged rats were used to study the distribution of surfactant proteins in Clara cells and type II pneumocytes. Serial sections of paraffin embedded lung tissue were sequentially immunostained by the avidin-biotin-peroxidase complex (ABC) technique. Antisera against SP-A, SP-B and Clara cell specific protein (CC10) were used. We determined stereologically' the surface fraction of immunolabelled cells and semiquantitatively the percentage of test fields containing labelled alveolar macrophages. In allergen sensitized and provocated rat lungs: (1) the surface fraction of SP-A and SP-B positive Clara cells was significantly reduced, (2) the surface fraction of Clara cells stained with CC10 was coincided with controls, (3) the surface fraction of SP-A and not of SP-B possitive type II pneumocytes decreased significantly, (4) a significantly higher percentage of test fields with SP-A labelled alveolar macrophages was evaluated. Thus, in this animal model of asthma the inflammatory process after allergen challenge is accompanied by alterations in the distribution patterns of SP in Clara cells and type II pneumocytes.

Optical coherence tomography (OCT) is an elegant technology for imaging of tissues and organs and has been established for clinical use for around a decade. Thus, it is used in vivo but can also serve as a valuable ex vivo imaging tool in experimental research. Here, a brief overview is given with a focus on an ex vivo application of OCT. Image and video examples of freshly obtained murine lungs are included. The main advantage of OCT for ex vivo analysis is the non-contact, non-invasive, and non-destructive fast acquisition of a three-dimensional data set with micrometer-resolution.

The phosphodiesterase 4 inhibitor cilomilast demonstrated strong inhibitory effects in a model of allergic contact dermatitis. In this study, we examined whether this inhibitory effect is at least partly due to modulation of dendritic cell function. Bone marrow-derived dendritic cells were pulsed with the sensitizer toluene-2,4-diisocyanate and administered subcutaneously to nonsensitized mice. Five days later, the mice were challenged with a low dose of toluene-2,4-diisocyanate onto the ears. In contrast to sham-treated mice, mice obtaining toluene-2,4-diisocyanate pulsed dendritic cells showed a significant increase in ear swelling. This swelling was not influenced when the dendritic cells were pre-incubated with cilomilast. When cilomilast was administered systemically simultaneously to the application of toluene-2,4-diisocyanate pulsed cells, there was an impaired allergic reaction provoked 5 days later. Additionally, a topical treatment with cilomilast resulted in a significant inhibition of skin dendritic cell migration. These results indicate that the antigen-presenting function of dendritic cells is not influenced by cilomilast but the dendritic cell T cell interaction and dendritic cell migration is modulated.

To investigate, whether T lymphocytes alone are sufficient to induce glomerulonephritis, a model in SCID mice was developed. Conditions for the generation and exclusive glomerular targeting of crosslinked ovalbumin (OVA) polymers and a series of OVA-specific T-cell clones and lines were established. Only a well-defined subfraction of OVA polymers exclusively targeted to the glomerular mesangium without causing local alteration in the absence of IgG. From numerous T-cell preparations spanning different Th-1/-2 profiles one T-helper cell clone characterized by ELISPOT assay as pure Th-1 (IFN-γ and IL-2) induced nephritislike pathology. Histological examination at days 1, 2, 5, and 21 showed major infiltrates in proximal tubular regions (PTR) at day 5 accompanied by significant proteinuria. No injury was observed after deposition of irrelevant antigen or injection of other T-cell preparations. Detailed histological analysis revealed that Th-1 cell numbers peaked early in glomeruli (2.1 ± 0.6 vs 0/gcs). Macrophages, however, were hardly detectable in glomeruli (0.5 ± 0.3/gcs) at this time, while they formed the major constituent of the PTR infiltrates at day 5 (83 ± 1). These data in a new SCID nephritis model indicate that memory Th-1 cells together with localized antigen presenting cells trigger nephritis.

In allergic and inflammatory immune reactions of the respiratory tract, leukocytes migrate into the different compartments of the lung. The air space can easily be sampled by means of bronchoalveolar lavage. However, the subset composition in the bronchial wall or the lung interstitium often differs considerably from that of the bronchoalveolar lavage fluid. A further compartment involved in very heterogeneous immune reactions in the lung has thus far not been mentioned: the periarterial space. In numerous experiments in different species with virus, bacteria, fungi, or allergens, there was not only a leukocyte infiltration of the bronchial lamina propria but also infiltration around branches of the pulmonary artery. This thus far neglected compartment consists of a different type of capillary. Thus it is important not to overlook this area in studies on allergic or inflammatory immune reactions of the lung.

Summary Introduction: To establish primary osteoblast cultures is a challenge. The methods for isolation mostly comprise digestion with extracellular matrix degrading enzymes after mincing the bone samples. These methods are labour intensive and lead to an inefficient recovery of cells. Therefore, the aim of this study was to develop a more reliable method for culturing human osteoblasts. Materials and methods: Bone tissue specimens were obtained from 20 patients undergoing reconstructive operations. Bone specimens were dissected and put into petri dishes with the bottom covered with fibrin glue. To identify the nature of the outgrowing cells, cytological staining was performed, i.e. Von Kossa, Azan, Dahl's, alkaline phosphatase, and collagen type I. Results: Mean time interval of cellular outgrowth was 12 days after preparing the bone tissue specimens. Confluence of the cell cultures was reached after four to five weeks on average. All cells were positively stained using Von Kossa, alkaline Phosphatase and collagen type I. The matrix consisted of lime, calcium and collagens. Conclusion: A simplified method to culture osteoblasts from all kinds of bone tissue specimens is presented. The fibrin glue allows firm adhesion of the specimens to the petri dish. This allows the cells to grow out without disturbance. Normally, due to movements during medium exchange the adhesive bonds are disrupted. The fibrin glue retains the adhesive bonds. This method allows studying human osteoblasts in different clinical settings.

Summary Many investigations have been performed in characterising experimental bacterial infections in the lung of mice using several pathogens. Robust experimental pulmonary infection models require a reproducible method of application with defined numbers of pathogens to the respiratory tract without contaminating extrapulmonary tissues. At the same time trauma due to the experimental procedure should be kept to a minimum. So far several routes of administration have been used but a systematic comparison of these methods is still missing. Here we provide a comprehensive evaluation of view controlled i.t. instillation, tracheotomy, intranasal application, blind instillation and aerosol infection. An infection dose of up to 5 × 10 4 bacteria ( L. monocytogenes ) was applied to a group of ten mice by each technique and the animals were killed after 1h or 24h. The number of viable bacteria was estimated by plating homogenates of the lungs and spleens. In addition, pathological effects on lung tissue were examined by histology 24h after infection. The highest reproducibility was achieved after applying Listeria directly in the trachea under view or by tracheotomy. However, mice were severely affected in their vitality after tracheotomy. Thus, for topical application of bacterial suspension into the lung the view controlled i.t. instillation is most appropriate.

Summary In pulmonary immune reactions the cells which can be obtained by bronchoalveolar lavage (BAL) are only one part of the picture. In this study the kinetics of an experimental pulmonary immune response were investigated simultaneously in different lung compartments in the same rat. On days 0, 1, 2, 3, 4, 5 and 11 after intratracheal challenge with sheep red blood cells, leukocytes were taken from the bronchoalveolar, the interstitial and the marginal lung vascular pool as well as from the peripheral blood. Total numbers of granulocytes, NK cells, B and T cells, CD4 + and CD8 + cells were determined. Histology and in vivo labeling of proliferating cells was performed. On day 1 after challenge an increase of granulocytes in the BAL was found. In the BAL the total number of T lymphocytes increased on day 1 and day 2 and the CD4/CD8 ratio increased from day 1 to day 5, indicating an influx of CD4 + T cells. Changes in the lung interstitium showed a similar tendency, but were not found in the marginal pool or blood. Histologically cellular infiltrates were seen around the pulmonary small vessels. Little local proliferation occurred in the different lung compartments, indicating mainly immigration of cells. Further studies will focus on the expression of adhesion molecules during an immune response, to learn more about the mechanisms responsible for the increase of lymphocytes.

Centrally applied neuropeptide Y (NPY) interacts with the autonomic nervous system and the hypothalamo-pituitary-adrenal (HPA) axis activity. Since these physiological systems have been shown to modulate innate immune functions, the effects of intracerebroventricular (i.c.v.) NPY administration on leukocyte subsets in the blood, spleen and intravascular pool of the lung, blood granulocyte chemiluminescence response, and splenic natural killer (NK) cell-mediated lysis were studied in Lewis rats. Concentration-dependent NPY effects were tested at 15 min and 24 h post i.c.v. injection at dosages of 10(-6) M, 10(-9) M, and 10(-12) M. Time dependent effects were investigated at 15 min, 1 h and 24 h after i.c.v. administration of 10(-9) M NPY. Compared to saline controls, an increased number of granulocytes and NK cells in the blood, associated with a decreased granulocyte function and NK cytotoxicity was observed 15 min following NPY infusion. This initial immunosuppression was followed by long lasting stimulatory effects of NPY on the functional capacity of both cell populations when tested at 1 h and 24 h. The dosage of i.c.v. 10(-6) M NPY produced no changes, whilst 10(-9) M produced maximal, and 10(-12) M still significant effects. Results provide evidence that centrally applied NPY influences innate immunity in a dose and time dependent fashion. Cell mobilization from the vascular marginal pool is likely to be an underlying mechanism for the initial immunosuppression.

Fragestellung Knorpel- und Knochendegeneration stellen ein groses volkswirtschaftliches und individualspezifisches Problem dar, mit deren Losung sich die Methoden der regenerativen Medizin befassen. Das Ziel dieser Studie war es, den Einfluss von einem chondrogenen Differenzierungsmedium und/oder einer mechanischen Stimulation auf Primarstabilitat und morphologischen Aufbau eines osteochondralen Transplantates zu untersuchen. Methodik Wahrend unfallchirurgischen Eingriffen wurden stromale Zellen aus dem Knochenmark (BMSCs) des Beckenkamms gewonnen, die isoliert, passagiert und fur sieben Tage mithilfe eines u.a. TGF-β2- und IGF-1-haltigen Differenzierungsmediums in zweidimensionaler Kultur chondrogen vordifferenziert wurden. Anschliesend erfolgte der Transfer in eine biologische Hybridmatrix (CaReS Gel, Arthrokinetics, Esslingen, Germany und Tutobone, Tutogen Medical GmbH, Neunkirchen a. Br., Germany), die in einer speziell entwickelten Glasapparatur fur 24 h durch eine Kombination von Druck- und Zugkraften komprimiert wurde. Die so entstandenen osteochondralen Konstrukte wurden verschiedenen Stimulationsgruppen zugeteilt, die sich in Stimulationsdauer, Stimulationsart (dynamisch im Bioreaktor oder statisch) und Anwesenheit der Wachstumsfaktoren unterschieden. Die Auswertung erfolgte dreigliedrig in Nasschemie (DNA- und GAG-Gehalt), Histologie (immunhistochemische Bestimmung der Matrixbestandteile) und Biomechanik, bei der unter Bedingungen der constraint compression die Steifigkeit errechnet wurde. Ergebnisse Histologisch zeigte sich eine homogene und vitale Zellverteilung (Live/Dead Assay: >70% vitale Zellen). Die noch ausstehenden immunhistochemischen Ergebnisse werden genauere Informationen uber die Morphologie der Konstrukte liefern. Mit der biomechanischen Auswertung konnte ein statistisch signifikanter Anstieg (p Die statisch stimulierten Konstrukte wiesen nach drei und vier Wochen einen signifikant (p Schlussfolgerungen Bei der Erstellung eines Protokolls fur die Produktion eines osteochondralen Konstruktes mit Hilfe von humanen mesenchymalen Stammzellen in einer Hybridmatrix stellte sich die mechanische Stimulation als vorteilhaft in Hinblick auf die mechanische Stabilitat der Konstrukte heraus. Obwohl Wachstumsfaktoren einen bekannten Stellenwert in der chondrogenen Zelldifferenzierung besitzen, konnte in dieser Studie kein signifikanter Einfluss der Hinzunahme von Mediumzusatzen gezeigt werden. Das Herauslassen dieser Faktoren aus dem Protokoll sowie eine Verkurzung der Stimulationsdauer kann diskutiert werden, um Vorteile bezuglich der Kosten und der klinischen Anwendbarkeit zu erzielen.

Allergic diseases like bronchial asthma are increasing in societies with western lifestyle. In the last years substantial progress was made in the understanding of the underlying mechanisms and explanations like the hygiene hypothesis were developed. However the exact mechanisms of the physiological and immunological events in the lung leading to bronchial asthma are still not fully understood. Therefore, animal models of asthma have been established and improved to study the complex cellular interactions in vivo. Since mice became the most frequently used animal species the methods for detecting lung physiology, e.g. lung function measurements were adapted to the small size of the murine lung. Laser-dissection and precision cut lung slices have become common techniques to get a view into distinct lung compartments and cells. In addition genomic and proteomic approaches are now used widely. On the other hand a major conclusion of the workshop stated that more than one species is necessary in research and for pharmacological screening in asthma and COPD. The resulting new understanding in the mechanisms of asthma pathogenesis has lead to a rapid identification of novel pharmaceutical targets for treatment of the disease.

model, focusing on relevant signaling cascades. Rodent left lungs were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received 10 mg/kg of intravenous MMF 90 minutes prior to ischemia. Lung injury was quantitated utilizing neutrophil accumulation (MPO content), vascular permeability to radiolabeled albumin, and bronchoalveolar lavage (BAL) inflammatory cell counts. Left lungs were also analyzed for transcription factor activation by EMSAs with supershifts, and BAL proinflammatory mediator protein content by ELISA. MMF significantly reduced lung vascular permeability indices (0.45 vs. 0.85, p 0.001), MPO content (0.26 vs. 0.37, p 0.04), and alveolar leukocyte counts (170 vs. 209 x 10 cells, p 0.03) at 4 hrs of reperfusion. At 15 min of reperfusion, there was significant attenuation of AP-1 (p 0.015) and EGR-1 (p 0.001) activation, while NFkB was unaffected (p 0.3). Reductions in BAL MCP-1 (1194 vs. 597 ng/ml, p 0.01) and CINC (1462 vs. 945 pg/ml, p 0.03) protein content were found at 4 hrs of reperfusion. MMF limits lung vascular permeability and inflammatory cell recruitment in a rodent LIRI model. The protection appears to be mediated in part via attenuated EGR-1 and AP-1-dependent transcriptional activation of proinflammatory chemokines.