Lars Bräuer

Virtual microscopy—The future of teaching histology in the medical curriculum?

Conventional continuing education in microscopic anatomy, histopathology, hematology and microbiology has hitherto been carried out using numerous sets of sectioned tissue specimens in a microscopy laboratory. In comparison, after digitalization of the sections it would be possible to access teaching specimens via virtual microscopy and the internet at any time and place. This would make it possible to put innumerable new learning scenarios into practice. The present article elucidates the advantages of virtual microscopy in histology instruction and presents a concept of how virtual microscopy could be introduced into the teaching of microscopic anatomy in several steps. Initially, the presently existing microscopic teaching specimens would be digitalized and made available on-line without restriction. In a second step, instruction would be shifted to an emphasis on virtual microscopy, utilizing all of the advantages offered by the technique. In a third step, the microscopic contents could be networked with other anatomical, radiological and clinical content on-line, thus opening new learning perspectives for students of human and dental medicine as well as those of medically related courses of study. The advantages and disadvantages of such a concept as well as some possibly arising consequences are discussed in the following.

Detection and Localization of the Hydrophobic Surfactant Proteins B and C in Human Tear Fluid and the Human Lacrimal System

Purpose: To evaluate the expression and presence of the surfactant proteins (SP) B and C in the lacrimal apparatus at the ocular surface and in tear fluid. Methods: Expression of SP-B and SP-C was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, meibomian gland, accessory lacrimal glands, cornea, and nasolacrimal ducts. The deposition of the hydrophobic proteins SP-B and SP-C was determined by Western blot and immunohistochemistry in healthy tissues, tear fluid, and aqueous humor. Results: The presence of both SP-B and SP-C on mRNA and protein level was evidenced in healthy human lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts. Moreover, both proteins were present in tear fluid but were absent in aqueous humor. Immunohistochemical investigations revealed production of both peptides by acinar epithelial cells of the lacrimal gland and additionally by accessory lacrimal glands of the eyelid as well as epithelial cells of the conjunctiva and nasolacrimal ducts. Immunohistochemically, healthy cornea and goblet cells revealed no reactivity. Conclusions: Besides the recently detected surfactant-associated proteins SP-A and SP-D, our results show that SP-B and SP-C are also peptides of the tear film, the ocular surface, and the lacrimal apparatus. Based on the current knowledge of lowering surface tension in alveolar lung cells, a similar effect of SP-B and SP-C may be assumed concerning the tear film.

Schirmer strip vs. capillary tube method: Non-invasive methods of obtaining proteins from tear fluid

Human tear fluid is a complex mixture containing over 500 solute proteins, lipids, electrolytes, mucins, metabolites, hormones and desquamated epithelial cells as well as foreign substances from the ambient air. Little is known to date about the function of most tear components. The efficient and gentle collection of tear fluid facilitates closer investigation of these matters. The objective of the present paper was to compare two commonly used methods of obtaining tear fluid, the capillary tube and Schirmer strip methods, in terms of usefulness in molecular biological investigation of tear film. The comparative protein identification methods Bradford and Western Blot were used in the analyses to this end. The surfactant proteins (SP) A-D recently described as present on the eye surface were selected as the model proteins. Both methods feature sufficient uptake efficiency for proteins in or extraction from the sampling means used (capillary tube/Schirmer strip). The total protein concentration can be determined and the proteins in the tears can be detected - besides the hydrophilic SP-A and D also the non-water-soluble proteins of smaller size such as SP-B and C. Thus both methods afford a suitable basis for comparative analysis of the physiological processes in the tear fluid of healthy and diseased subjects. On the whole, the Schirmer strip has several advantages over the capillary tube.

Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse β-defensins-2, -3 and -4 (mBD2–4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that β-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.

Human parotid and submandibular glands express and secrete surfactant proteins A, B, C and D

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.

Strategies against burnout and anxiety in medical education--implementation and evaluation of a new course on relaxation techniques (Relacs) for medical students.

Burnout and stress-related mental disorders (depression, anxiety) occur in medical students and physicians with a significantly higher prevalence than in the general population. At the same time, the learning of coping mechanisms against stress is still not an integral part of medical education. In this pilot study we developed an elective course for learning relaxation techniques and examined the condition of the students before and after the course. 42 students participated in the semester courses in 2012 and 2013 as well as in a survey at the start and end of each course. The students were instructed in autogenic training (AT) and progressive muscle relaxation according to Jacobsen (PMR) with the goal of independent and regular exercising. At the beginning and the end of the semester/course the students were interviewed using standardized, validated questionnaires on burnout (BOSS-II) and anxiety (STAI-G), depression (BDI), quality of life (SF-12) and sense of coherence (SOC-L9). We compared the results of our students participating in Relacs with results from eight semester medical students (n = 88), assessed with the same questionnaires at similar points of time within their semester. Participating students showed a significant decline in cognitive and emotional burnout stress and in trait anxiety. Furthermore, they showed a reduction in state anxiety and a conspicuous decrease in mean depression. The sense of coherence increased at the same time. A comparative cohort of medical students of 8th semester students, showed lower values for the specified measurement parameters at the beginning, but showed no progressive changes. Our course introducing AT and PMR led to a significant reduction of burnout and anxiety within the participating group of medical students. Even the course attendance for just one semester resulted in significant improvements in the evaluated parameters in contrast to those students who did not attend the course.

Detection of surfactant proteins A, B, C, and D in human nasal mucosa and their regulation in chronic rhinosinusitis with polyps.

Backround The nasal mucosa is characterized by a multirow high prismatic ciliated epithelium representing the first barrier of the immune defense system against microbial and other environmental pathogenic influences. A number of nonspecific defense mechanisms, including the presence of lactoferrin, peroxidases, proteases, interferons, and lysozymes in nasal secretions, act to counter inflammatory processes. The surfactant proteins (SPs) known from the lungs are important components of the innate immune system. They also influence the rheology of fluids and reduce the surface tension of gas–fluid interphases. The objective of this study was to investigate the protein expression of all four SPs. A specific aim was detection and characterization of SP-C, which had previously not been confirmed in human nasal mucosa. Methods The expression of mRNA for SP-A, -B, -C and -D was investigated using reverse transcriptase polymerase chain reaction on samples of both healthy nasal mucosa and nasal mucosa altered by inflammatory processes (allergic rhinitis and chronic rhinosinusitis). The distribution of all four proteins was determined with monoclonal antibodies using Western blot analysis as well as immunohistochemical methods. Results The results show that all four SPs, including SP-C not detected before this, are nasal mucosa components. A shift was also observed in the expression behavior of the SP-A, -B, and -D in nasal mucosa with inflammatory changes. Conclusion Based on these results, SPs appear to have an important function in immunologic and rheological process of the nasal mucosa and support the prospective therapeutic use of liposomal nasal sprays.

Regulation of MUC16 by inflammatory mediators in ocular surface epithelial cell lines

The aim of the present study was to evaluate the regulation of membrane-anchored mucin MUC16 by proinflammatory cytokines and bacterial components at the ocular surface. Expression and distribution of MUC16 in conjunctival (HCjE) and corneal (HCE) epithelial cell lines was monitored by RT-PCR and immunohistochemistry. To determine the regulation of MUC16, cultured HCjEs and HCEs were stimulated with different cytokines, bacterial components and bacterial supernatants, and analyzed by real-time PCR, immunodot blot and immunohistochemistry. The results indicate that MUC16 is differentially regulated between HCjEs and HCEs after challenge with inflammatory mediators and suggest shedding of MUC16 from the ocular surface epithelia into the tear film. This seems to be precisely regulated. MUC16 shedding can be differentially increased and decreased, suggesting a protective function of membrane-anchored MUC16 and supporting the hypothesis that dysregulation of membrane-anchored MUC16 at the ocular surface may be involved in dry eye pathology.

MUC16 in the lacrimal apparatus

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).

The Detection of Surfactant Proteins A, B, C and D in the Human Brain and Their Regulation in Cerebral Infarction, Autoimmune Conditions and Infections of the CNS

Surfactant proteins (SP) have been studied intensively in the respiratory system. Surfactant protein A and surfactant protein D are proteins belonging to the family of collectins each playing a major role in the innate immune system. The ability of surfactant protein A and surfactant protein D to bind various pathogens and facilitate their elimination has been described in a vast number of studies. Surfactant proteins are very important in modulating the hosts inflammatory response and participate in the clearance of apoptotic cells. Surfactant protein B and surfactant protein C are proteins responsible for lowering the surface tension in the lungs. The aim of this study was an investigation of expression of surfactant proteins in the central nervous system to assess their specific distribution patterns. The second aim was to quantify surfactant proteins in cerebrospinal fluid of healthy subjects compared to patients suffering from different neuropathologies. The expression of mRNA for the surfactant proteins was analyzed with RT-PCR done with samples from different parts of the human brain. The production of the surfactant proteins in the brain was verified using immunohistochemistry and Western blot. The concentrations of the surfactant proteins in cerebrospinal fluid from healthy subjects and patients suffering from neuropathologic conditions were quantified using ELISA. Our results revealed that surfactant proteins are present in the central nervous system and that the concentrations of one or more surfactant proteins in healthy subjects differed significantly from those of patients affected by central autoimmune processes, CNS infections or cerebral infarction. Based on the localization of the surfactant proteins in the brain, their different levels in normal versus pathologic samples of cerebrospinal fluid and their well-known functions in the lungs, it appears that the surfactant proteins may play roles in host defense of the brain, facilitation of cerebrospinal fluid secretion and maintenance of the latters rheological properties.