Christina Hipler

Lectin‐Binding Sites in Normal Human Testis/Lektinbindungsstellen normaler humaner Hoden

Summary: Nine fluorescein isothiocyanate (FITC) — labelled lectins have been used to investigate the distribution of glycoconjugates in unfixed frozen and Bouin‐fixed sections of normal human testis.

Influences of nasal lavage collection-, processing- and storage methods on inflammatory markers--evaluation of a method for non-invasive sampling of epithelial lining fluid in cystic fibrosis and other respiratory diseases.

BACKGROUND Non-invasive sampling of airway epithelial-lining-fluid by nasal lavage (NL) is an emerging method to monitor allergy, infection and inflammation in patients with respiratory diseases. However, the influences of collection-, processing- and storage-methods have not been sufficiently evaluated and standardized. METHODS Influences of repeated NL, centrifugation setups, repeated freezing and thawing, and protease inhibitors on mediator concentration were evaluated in healthy controls and CF patients, which serve as a model for chronic bacterial infection and inflammation. Polymorphonuclear leukocyte elastase (NE)/myeloperoxidase (MPO)/interleukin (IL)-1/IL-6/IL-8 and tumour necrosis factor alpha (TNF) concentrations were measured using ELISA and Multiplex Bead-Arrays. RESULTS NL-repetition within 0.5-4h markedly decreased NE, IL-8 and MPO-concentrations for up to 70%. NL centrifugation up to 250×g for cellular differentiation did not significantly influence mediator concentration in native and processed NL fluid. NL freezing and thawing markedly decreased IL-8 and MPO concentrations by up to 50% while NE remained stable. In contrast to preceding reports, storing at -70°C for ≥5 years led to significantly reduced mediator concentrations in NL compared to contemporary analyses, being most pronounced for IL-1β, IL-6 and TNFa. Storing of samples in the presence of protease inhibitors led to an increase in marker concentration for IL-8 (+27%) and MPO (+15%) even after one year of storage. CONCLUSIONS NL is an easy and robust technique for inflammation monitoring of the upper airways. For the first time we have shown that diagnostic NL should be performed only once daily to get comparable results. Whereas NL-fluid can be stored unprocessed at -70°C for cytokine analysis over 1-2 years with protease inhibitors supporting stability, ≥5 years storage as well as repeated freezing and thawing should be avoided.

Tissue Expansion in Pig Skin – A Histochemical Approach

In the present study, a porcine model for controlled skin expansion was investigated to improve our understanding of epidermal and vascular responses following stretching. The model is of outstanding importance not only for the clinical use of tissue expansion but provides interesting data for skin physiology and oncology, too.

BCR-ABL- and Ras-independent activation of Raf as a novel mechanism of Imatinib resistance in CML

Although the BCR-ABL tyrosine kinase inhibitor Imatinib has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML), acquired drug resistance remains a common problem in CML therapy. Resistance often arises from second-line mutations in BCR-ABL or overexpression of the BCR-ABL protein but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Imatinib-resistant CML cell lines have been widely used for comparative proteome/genome-wide expression screens in order to decipher resistance mechanisms but a clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Imatinib has not yet evolved from those studies. Here, we report the identification of a novel mechanism for Imatinib resistance in CML cells with unaltered BCR-ABL function. Pharmacological analysis evidenced a constitutive, Imatinib-insensitive activation of the Erk-MAPK pathway in resistant cells. A systematic analysis of pathway constituents illustrated that Ras-GTP accumulation remained fully sensitive to Imatinib but c-Raf activity from serum-fed cultures was largely resistant to the drugs action. Sequencing excluded mutations in either B-Raf or c-Raf as the origin of resistance, indicating that a functional alteration in the regulation of c-Raf activity was responsible for this effect. Collectively, these findings highlight a novel mechanism of acquired Imatinib resistance based on the BCR-ABL and Ras-independent constitutive activation of the Erk-MAPK pathway through activated c-Raf, which could prove helpful for a better functional classification of the causes of Imatinib resistance in CML.

Distribution of glycoconjugates in human skin apperdages

The purpose of this paper is to describe the occurrence and distribution of glycoconjugates in normal human epidermis and skin appendages (pilosebaceous unit, eccrine sweat gland) by means of FITC-labelled lectins (ConA, WGA, UEA I). Both the outer hair root and the follicular ostium-epithel disclosed a glycoconjugate expression with close homology to interfollicular epidermis. The acinar epithelium of sebaceous glands and the inner layers of hair follicles showed a more or less distinct staining pattern. Lectin binding of eccrine sweat glands revealed marked differences between ducts and secretory coils. The epithelial distribution of glycoconjugates indicates a relatively independent differentiation pathway of eccrine sweat glands compared with other specialized epithelia of the human skin.

Histochemistry of the porcine pilosebaceous unit.

The present study describes lectin and immunoreactivity in the pilosebaceous unit of porcine skin. Complex carbohydrates of mucin and biantennary Man/Gluc types were distributed among hair follicle epithelia (hair root sheaths, cuticula, shaft, and shaft matrix). Sebaceous glands expressed biantennary Man/Gluc carbohydrates and GalNAc residues. The expression of simple-type and epidermis-like keratins was confirmed by immunohistochemistry with monoclonal antibodies. Filaggrin-positive cells were found in the keratinizing zone of Henles layer in anagen follicles. The innermost layer of the outer hair root sheath was stained with antibodies against the epidermal growth factor-receptor, keratin 10 and Ki67 antigen. The differences to humans were remarkably small.

Fulminant papulopustular tinea corporis caused by Trichophyton mentagrophytes.

Anja Bornkessel*, Mirjana Ziemer, Siegrid Yu, Christina Hipler and Peter Elsner Department of Dermatology and Allergology, Friedrich-Schiller-University, Erfurter Str. 35, D-07743 Jena, Germany, and Department of Dermatology, University of California, San Francisco, CA, USA. *E-mail: anja.bornkessel@med.uni-jena.de [The first two authors contributed equally to the writing of this paper.] Accepted July 19, 2004.

Monitoring Wound Healing by Multiphoton Tomography/Endoscopy

Certified clinical multiphoton tomographs are employed to perform rapid label-free high-resolution in vivo histology. Novel tomographs include a flexible 360° scan head attached to a mechano-optical arm for autofluorescence and SHG imaging as well as rigid two-photon GRIN microendoscope. Mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged with submicron resolution in human skin. The system was employed to study the healing of chronic wounds (venous leg ulcer) and acute wounds (curettage of actinic or seborrheic keratosis) on a subcellular level. Furthermore, a flexible sterile foil as interface between wound and focusing optic was tested.

Lectin-Binding Sites in Testis of Men with Acrosomeless Round-Headed Spermatozoa/Testikuläre Lektinbindungsstellen bei Männern mit akrosomenlosen Rundkopfspermatozoen

Summary: We report herein about lectin histochemistry of seminiferous epithelia in two infertile men with exlusely acrosomeless round‐headed spermatozoa. FITC‐conjugated lectins (ConA, PNA, RCA II, WGA) have been employed on tissue sections of Bouin fixed testicular biopsies. RCA II gave a dot‐like fluorescence of the acrosomal region and WGA gave a cap‐like acrosomal fluorescence of spermatids. PNA — a marker of acrosomal differentiation — failed to stain spermatids. The binding of ConA to germ cells was not influenced by this syndrome. In conclusion, the syndrome of acrosomeless round‐headed spermatozoa is associated with selective perturbations of testicular lectin‐binding sites. They might contribute of the inability of sperm cells to adhere to and penetrate into ova.

FITC-insulin binding to normal and psoriatic epidermis.

FITC-insulin was prepared and applied to frozen human skin sections. In normal and nonlesional psoriatic epidermis, the binding was cytoplasmic in suprabasal cells. In psoriatic lesions, fairly stained or even unstained cells were littered in suprabasal epidermis indicating the expansion of selected basal cell qualities into the stratum spinosum.