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A first tricolor fluorescent pH nanosensor is presented, which was rationally designed from biocompatible carboxylated polystyrene nanoparticles and two analyte-responsive molecular fluorophores. Its fabrication involved particle staining with a blue-red-emissive dyad, consisting of a rhodamine moiety responsive to acidic pH values and a pH-inert quinoline fluorophore, followed by the covalent attachment of a fluorescein dye to the particle surface that signals neutral and basic pH values with a green fluorescence. These sensor particles change their fluorescence from blue to red and green, depending on the pH and excitation wavelength, and enable ratiometric pH measurements in the pH range of 3.0-9.0. The localization of the different sensor dyes in the particle core and at the particle surface was confirmed with fluorescence microscopy utilizing analogously prepared polystyrene microparticles. To show the application potential of these polystyrene-based multicolor sensor particles, fluorescence microscopy studies with a human A549 cell line were performed, which revealed the cellular uptake of the pH nanosensor and the differently colored emissions in different cell organelles, that is, compartments of the endosomal-lysosomal pathway. Our results demonstrate the underexplored potential of biocompatible polystyrene particles for multicolor and multianalyte sensing and bioimaging utilizing hydrophobic and/or hydrophilic stimuli-responsive luminophores.

Fluorescent binders of the estrogen receptor (ER) are used in binding assays and in detection or imaging studies. However, fluorescence labelling of ER ligands usually leads to substantial decreases in binding affinity. In this study, we describe the development of high affinity fluorescent ER ligands. Cyanine dyes of the MiDye series were directly attached to the SERMs 4-hydroxytamoxifen (OHT) and raloxifene (Ral); linkers were deliberately omitted. This approach yielded conjugates with ERα binding affinities superior to the natural ligand estradiol. The OHT- and Ral-MiDye conjugates emitted in the 600–800 nm range. First round staining experiments showed that the conjugates, but not the dyes alone, accumulate in cells expressing estrogen-binding receptors.

Polyglycerol based nanogels (nPG) can function as cellular delivery systems. These nPGs are synthesized with different amine densities (nPG amines) by acid-catalyzed epoxide-opening polymerization using a mini-emulsion approach and surface modification. All the synthesized nanogels are characterized by NMR, dynamic light scattering, and ζ-potential, showing slightly positive surface charge and a homogeneous size of ≈100 nm. The use of these systems for delivery applications is demonstrated with regard to polyplex formation, cytotoxicity, and cellular uptake studies. It is depicted that the CE50 value of the high loaded nPG amines is eight times higher than the low loaded ones. The influence of the amine loading percentage on the nanogel and the effects of polyvalency in these architecture is discussed.

In this study, the extent to which the scaffold architecture of polyglycerol sulfates affects inflammatory processes and hemocompatibility is investigated. Competitive L-selectin binding assays, cellular uptake studies, and blood compatibility readouts are done to evaluate distinct biological properties. Fully glycerol based hyperbranched polyglycerol architectures are obtained by either homopolymerization of glycidol (60% branching) or a new copolymerization strategy of glycidol with ethoxyethyl glycidyl ether. Two polyglycerols with 24 and 42% degree of branching (DB) are synthesized by using different monomer feed ratios. A perfectly branched polyglycerol dendrimer is synthesized according to an iterative two-step protocol based on allylation of the alcohol and subsequent catalytic dihydroxylation. All the polyglycerol sulfates are synthesized with a comparable molecular weight and degree of sulfation. The DB make the different polymer conjugates perform different ways. The optimal DB is 60% in all biological assays.

The development of effective polymer-based nanocarriers which are able to target diseased tissues still remains a great challenge in current research. Dendritic polyglycerols have emerged as novel polymeric scaffolds that have demonstrated a great potential for diverse biomedical applications. These architectures have already proven their usefulness in therapeutic approaches related to multivalency, given by the synergy between the nanosized dimensions combined with the high density of functional groups. However, a continuous effort is necessary to modify and tailor polyglycerol architectures to fit the future demands of biomedical applications. The present work deals with the development of a general synthetic strategy that allows the linkage of low molecular weight dendritic polyglycerols to fluorescent dyes and cell targeting ligands. The receptor mediated cellular uptake of the polyglycerol conjugates highlight their potential to acts as new targeted nanocarriers that should be able to decrease non-specific cellular uptake.

Herein we describe a platform technology for the synthesis and characterization of partially aminated, (35)S-labeled, dendritic polyglycerol sulfate (dPG(35)S amine) and fluorescent dPGS indocarbocyanine (ICC) dye conjugates. These polymer conjugates, based on a biocompatible dendritic polyglycerol scaffold, exhibit a high affinity to inflamed tissue in vivo and represent promising candidates for therapeutic and diagnostic applications. By utilizing a one-step sequential copolymerization approach, dendritic polyglycerol (Mn ≈ 4.5 kDa) containing 9.4% N-phthalimide protected amine functionalities was prepared on a large scale. Sulfation and simultaneous radio labeling with (35)SO3 pyridine complex, followed by cleavage of the N-phthalimide protecting groups, yielded dPG(35)S amine as a beta emitting, inflammation specific probe with free amino functionalities for conjugation. Furthermore, efficient labeling procedures with ICC via iminothiolane modification and subsequent "Michael" addition of the maleimide functionalized ICC dye, as well as by amide formation via NHS derivatized ICC on a dPGS amine scaffold, are described. The dPGS-ICC conjugates were investigated with respect to their photophysical properties, and both the radiolabeled and fluorescent compounds were comparatively visualized in histological tissue sections (radio detection and fluorescence microscopy) of animals treated with dPGS. Furthermore, cellular uptake of dPGS-ICC was found in endothelial cord blood (HUVEC) and the epithelial lung cells (A549). The presented synthetic routes allow a reproducible, controlled synthesis of dPGS amine on kilogram scale applying a one-pot batch reaction process. dPGS amine can be used for analysis via radioactivity or fluorescence, thereby creating a new platform for inflammation specific, multimodal imaging purposes using other attachable probes or contrast agents.

The rational design of bright optical probes and dye-biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye-protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye-dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M(-1) cm(-1) and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS.

Microscopical visualization of nanoparticles in tissues is essential for assessing their distribution in whole organisms and their interaction with the cellular microenvironment, including possible toxic effects. However, labeling of nanoparticles with fluorescent dyes may affect their physicochemical properties. Moreover, the detection of organic nanoparticles in their tissue context often poses a particular challenge due to their closer similarities with biomolecules. As part of a biodistribution and toxicity study on organic anti-inflammatory nanoscaled dendritic polyglycerol sulfate amine (dPGS amine) we have established light microscopic autoradiography (LMA) for the tracking of 35 S labeled dPGS in standard histopathological tissue samples following intravenous injection in mice. The dPG 35 S amine was specifically localized in hepatic Kupffer cells with no histopathologic evidence of toxic, degenerate or inflammatory side effects. The combination of radiolabeling of organic nanoparticles with LMA offers a novel approach for their localization in microscopical slides, also allowing for a simultaneous standard toxicopathology analysis. From the Clinical Editor In this study, a novel light microscopic autoradiography utilizing 35 S isotope demonstrates a combined approach to visualize nanoparticle locations in microscopic slides with no obvious toxicity to the studied cells and with minimal external hazard.

The destruction of articular cartilage is a critical feature in joint diseases. An approach to selectively target the damaged tissue is promising for the development of diagnostic and therapeutic agents. We herein present the interaction of dendritic polyglycerol (dPG) anions with native and inflamed cartilage. Confocal laser scanning microscopy revealed the inert character of dPG and low functionalized dPG bisphosphonate (dPGBP7%) toward cartilage in vitro. An enhanced binding was observed for highly functionalized dPG bisphosphonate, sulfate, and phosphate, which additionally showed a higher affinity to IL-1β treated tissue. The mixed anion containing sulfate and bisphosphonate groups exhibited an exceptionally high affinity to cartilage and strongly bound to collagen type II, as shown by a normalized fluorescence-based binding assay. All polyglycerol anions, except dPGBP7%, were taken up by chondrocytes within 24 h and no cytotoxicity was found up to 10−5 M. In a rheumatoid arthritis model, dPGBP7% accumulated in mineralized compartments of inflamed joints and showed an increasing affinity to cartilage with higher clinical scores, as evident from histological examinations. For dPGS no interaction with bone but a strong binding to cartilage, independent of the score, was demonstrated. These results make dPG anions promising candidates for the selective targeting of cartilage tissue.

A water-soluble molecular transporter with a dendritic core-shell nanostructure has been prepared by a tandem coordination, ring-opening, hyperbranched polymerization process. Consisting of hydrophilic hyperbranched polyglycerol shell grafted from hydrophobic dendritic polyethylene core, the transporter has a molecular weight of 951 kg/mol and a hydrodynamic diameter of 17.5 ± 0.9 nm, as determined by static and dynamic light scattering, respectively. Based on evidence from fluorescence spectroscopy, light scattering, and electron microscopy, the core-shell copolymer transports the hydrophobic guests pyrene and Nile red by a unimolecular transport mechanism. Furthermore, it was shown that the core-shell copolymer effectively transports the hydrophobic dye Nile red into living cells under extremely high and biologically relevant dilution conditions, which is in sharp contrast to a small molecule amphiphile. These results suggest potential applicability of such core-shell molecular transporters in the administration of poorly water-soluble drugs.

Die NeuroMastER-Arbeiten untersuchen die Immunerkrankung Bulloses Pemphigoid (BP) bezuglich grundlegender pathophysiologischer Aspekte von Umwelteinflussen. Nachdem in den vorangegangenen Arbeiten vor allem Lokalisationsstudien bezuglich des VPAC2-Rezeptors fur Vasoaktives Intestinales Polypeptid (VIP) erfolgten, war es das Ziel der vorliegenden Studie, quantitative Aspekte der Expression dieses Rezeptors festzustellen. Mittels immunhistochemischer Verfahren wurden dabei auf translationeller Ebene Unterschiede in der Mastzell-spezifischen VPAC2-Expression unter den pathophysiologischen Bedingungen des Bullosen Pemphigoids festgestellt.

Mastzellen und ihre Mediatoren sind wichtige Bestandteile des neuroimmunokutanen Systems und spielen eine entscheidende Rolle bei Interaktionen des Organismus mit der Umwelt. In der vorliegenden Arbeit wurde aufbauend auf vorangehenden NeuroMastER-Studien eine Quantifizierung Chymase- und Tryptase-exprimierender Mastzellen in normaler Haut und bei Bullosem Pemphigoid vorgenommen. Dabei wurden signifikante Unterschiede festgestellt, die auf eine essentielle Rolle der Mediatoren bzw. des Zelltyps hinweisen. Zukunftige Studien mussen die genauen Effekte der Umwelteinflusse bei Bullosem Pemphigoid charakterisieren.

Mastzellen spielen an den Grenzflachen des Organismus eine wesentliche Rolle bei der Reaktion auf Umwelteinflusse. Diese Umwelteinflusse werden durch Neuropeptide und ihre Rezeptoren vermittelt. Ein Kandidatenrezeptor fur die regulatorische Steuerung des Gefastonus bei entzundlichen Hauterkrankungen ist der VPAC2-Rezeptor des Vasoaktiven Intestinalen Polypeptids. Im Rahmen der vorliegenden NeuroMAstER-Teilstudie wurde die Reaktivitat dieses Rezeptors in Mastzellen der normalen Haut mittels quantitativer Immunhistochemie und Serienschnitttechnik charakterisiert. Es zeigte sich ein signifikant hoherer Anteil VPAC2-exprimierender Zellen an der Gesamtmastzellzahl in den tieferen Schichten als in den oberen Schichten der gesunden Dermis. Zusammenfassend steuert diese NeuroMAstER-Teilstudie einen wichtigen Beitrag zur Erfassung der Bedeutung von VPAC2 unter normalen Bedingungen bei und offnet das Feld fur weitere Studien zur pathophysiologischen Bedeutung des Rezeptors.

Nachdem in einer vorangegangenen NeuroMastER-Studie eine Lokalisation der mRNA des V PAC2-Rezeptors fur Vasoaktives Intestinales Polypeptid (VIP) erfolgte, war es das Ziel der vorliegenden Studie, in situ Proteindaten zur Lokalisation von V PAC2 bei Bullosem Pemphigoid (BP) zu sammeln. Diese Immunerkrankung unterliegt einer Vielzahl von Umwelteinflussen.

Umwelteinflusse und umweltmedizinische Aspekte werden zunehmend bezuglich ihrer Rolle bei der Progression einer Vielzahl von Erkrankungen diskutiert. In diesem Rahmen soll das NeuroMastER-Projekt den Zusammenhang zwischen Neuromediatoren, Mastzellen und Umweltinteraktionen untersuchen. In der vorliegenden NeuroMastER-Teilstudie wurde die Expression der mRNA des VPAC2-Rezeptors fur Vasoaktives Intestinales Polypeptid (VIP) durch In-situ-Hybridisierung bei Bullosem Pemphigoid (BP) untersucht. Dabei wurden Signale fur VPAC2-mRNA sowohl in normalen als auch BP-Gewebe in folgenden Zellen gefunden: Keratinozyten, mononukleare Zellen und Mastzellen, Endothelzellen kleiner Gefase, Haarfollikelzellen, Zellen holokriner Talgdrusen und ekkriner Schweisdrusen sowie in der Muskulatur kleiner Arterien und in Zellen des M. erector pili. Die vorliegende Studie bildet somit die Grundlage fur eine Vielzahl zukunftiger Arbeiten, welche die umweltmedizinisch relevanten Interaktionen von Entzundungszellen mit Neuromediatoren und ihren Rezeptoren charakterisieren sollen. Diese Mechanismen konnen sowohl im Bereich der Diagnostik umwelt- und arbeitsbedingter Erkrankungen eine Rolle spielen als auch in der Tertiarpravention.

To study the mechanism of cellular internalization, hyperbranched polyether derivatives consisting of amino-bearing hyperbranched polyglycerols (HPGs) of varied molecular mass and size range are designed and synthesized. HPGs were further fluorescently labelled by conjugating maleimido indocarbocyanine dye (ICC-mal). The conjugates are characterized by UV-vis spectroscopy, fluorescence profile, zeta potential, and dynamic light scattering. The uptake mechanism is studied by fluorescence-activated cell sorting (FACS) analysis, fluorescence spectroscopy, and confocal microscopy with human lung cancer cells A549, human epidermoid carcinoma cells A431, and human umbilical vein endothelial cells (HUVEC) cells. For the first time, the results suggest that the higher-molecular-weight HPGs (40-870 kDa) predominantly accumulate in the cytoplasm much better than their low-molecular-weight counterparts (2-20 kDa). The HPG nanocarriers discussed here have many biomedical implications, particularly for delivering drugs to the targeted site.

Mast cells have recently been related to nonallergic chronic organ damage and fibrosis. In the present study, we analyzed mast cell number, localization, and maturation in the kidney of a relatively unique group of middle-aged accident victims with primary essential hypertension and in normotensive controls ( n = 8 per group, Caucasians, predominantly male). Hypertensive kidneys showed a significantly higher degree of arteriolosclerosis. However, glomerular and tubulointerstitial matrix accumulation did not differ significantly to normotensive controls indicating a relatively early stage of hypertensive nephropathy. Using toluidine blue staining, renal mast cell number was found to be fivefold higher in hypertensive subjects compared with normotensive controls. Mast cells were primarily located in the peritubular interstitial spaces, some perivascular, but not in glomeruli. In a series of immunohistological staining studies, mast cell maturation grading showed that expression of early hematopoietic precursor cell marker CD34 did not differ between both groups. In contrast, mast cells were mostly positive for IgE receptor, tryptase, and chymase indicating a mature, differentiated cell phenotype in hypertensive nephropathy. Renal expression of stem cell factor was markedly upregulated in primary hypertension. Kidney macrophage and lymphocyte numbers were similar in both groups. In conclusion, human hypertensive kidney disease shows an early and conspicuous upregulation of stem cell factor along with an increased number of mature mast cells. The results suggest that renal mast cell accumulation may play a role in the pathogenesis of human hypertensive nephropathy.

Summary Background Neurotrophins such as nerve growth factor or brain-derived neurotrophic factor influence neuronal proliferation and differentiation via the low-affinity pan-neurotrophin receptor p75NTR that may play a pivotal role in linking the immune with the nervous system. Because the precise regulation of p75NTR gene transcription in mast cells under states of allergic inflammation has not been investigated in detail so far, the present studies assessed the gene regulation and expression of this receptor. Methods Transcriptional expression of p75NTR in human skin was studied in isolated cutaneous cells by means of RT-PCR. In situ lesional mast cell p75NTR expression was analysed by immunohistochemistry. Results The p75NTR mRNA expression was found in isolated human skin mast cells and keratinocytes. Lower mRNA levels were present in fibroblasts and melanocytes but no transcripts were found in endothelial cells. The p75NTR protein expression was found in situ in lesional and non-lesional mast cells. A significantly increased expression of p75NTR protein was found in atopic dermatitis lesional mast cells when compared with control mast cell expression (P

The role of adhesion molecules in the control of hair follicle (HF) morphogenesis, regression and cycling is still rather enigmatic. Since the adhesion molecules E- and P-cadherin (Ecad and Pcad) are functionally important, e.g. during embryonic pattern formation, we have studied their expression patterns during neonatal HF morphogenesis and cycling in C57/BL6 mice by immunohistology and semi-quantitative RT-PCR. The expression of both cadherins was strikingly hair cycle-dependent and restricted to distinct anatomical HF compartments. During HF morphogenesis, hair bud keratinocytes displayed strong Ecad and Pcad immunoreactivity (IR). While neonatal epidermis showed Ecad IR in all epidermal layers, Pcad IR was restricted to the basal layer. During later stages of HF morphogenesis and during anagen IV-VI of the adolescent murine hair cycle, the outer root sheath showed strong E- and Pcad IR. Instead, the outermost portion of the hair matrix and the inner root sheath displayed isolated Ecad IR, while the innermost portion of the hair matrix exhibited isolated Pcad IR. During telogen, all epidermal and follicular keratinocytes showed strong Ecad IR. This is in contrast to Pcad, whose IR was stringently restricted to matrix and secondary hair germ keratinocytes which are in closest proximity to the dermal papilla. These findings suggest that isolated or combined E- and/or Pcad expression is involved in follicular pattern formation by segregating HF keratinocytes into functionally distinct subpopulations; most notably, isolated Pcad expression may segregate those hair matrix keratinocytes into one functional epithelial tissue unit, which is particularly susceptible to growth control by dermal papilla-derived morphogens. The next challenge is to define which secreted agents implicated in hair growth control modulate these follicular cadherin expression patterns, and to define how these basic parameters of HF topobiology are altered during common hair growth disorders.

Background Anti-inflammatory nanoparticular compounds could represent a strategy to diminish osteoarthritis (OA) progression. The present study was undertaken to prove the uptake of nanoparticular dendritic polyglycerol sulfates (dPGS) by rat-derived articular chondrocytes and to answer the question of whether dPGS could modulate knee joint cartilage degradation in a rat OA model and whether complications could arise. Methods dPGS uptake and cytotoxicity was assessed in cultured primary rat-derived articular chondrocytes. Subsequently, OA was induced in the right knee joints of 12 male Wistar rats by medial collateral ligament and meniscus transection. Unoperated left knees remained as controls. Six weeks post surgery six rats were either treated daily (14 days) with 30 mg/kg dPGS (s.c.) or a similar volume of physiological saline. Animals were analyzed clinically for gait alterations. Explanted knee joints were studied histologically using OA scores according to Mankin (1971), Glasson et al., (2010) and the synovitis score according to Krenn et al., (2006). Liver, spleen and kidneys were analyzed for degenerative changes due to dPGS accumulation. Results dPGS was taken up after 2 hours by the chondrocytes. Whereas no significant clinical signs of OA could be detected, at the histological level, all operated rat knee joints revealed features of OA in the medial compartment. The values produced by both OA score systems were lower in rats treated with dPGS compared with saline-treated animals. Synovitis score did not significantly differ between the groups. The analyzed organs revealed no degenerative changes. Conclusions dPGS presented overall cyto- and biocompatibility, no accumulation in metabolizing organs and chondroprotective properties in the osteoarthritic knee joint. Electronic supplementary material The online version of this article (doi:10.1186/s12891-015-0844-3) contains supplementary material, which is available to authorized users.

Graft-versus-host disease (GvHD) is a severe immune reaction commonly occurring after hematopoietic stem cell transplantation. The outcome of patients who do not respond to the currently used immunosuppressive drugs is poor, thus there is an urgent need for the evaluation of new therapies. Heparin has a well-known anti-inflammatory effect and heparin analogues with a low anticoagulant effect are interesting candidates as new anti-inflammatory drugs. We explored the therapeutic potential of dendritic polyglycerol sulfates (dPGS), a novel class of heparin derivatives, on murine acute GvHD in vivo. The therapeutic effect of dPGS on murine GvHD was more intense after intravenous application compared to subcutaneous injection. An increased survival rate and improved clinical scores were observed in mice treated with 5 mg/kg once a week. In these animals, there was a reduction in the percentage of CD4(+) and CD8(+) T cells, which are the main effectors of GvHD. In addition, dPGS treatment decreased the number of tumor necrosis factor alpha (TNFα)-producing T cells. Increasing the dose of dPGS reversed the positive effect on survival as well as the clinical score, which indicates a small therapeutic range. Here, we report for the first time that dPGS have a significant immunosuppressive in vivo effect in a mouse model of severe acute GvHD. Therefore, we propose to study dPGS as promising candidates for the development of potential new drugs in the treatment of steroid-refractory GvHD patients first in larger animals and later in humans.

The synthesis of water-soluble dyes, which absorb and emit in the range between 650 and 950 nm and display high extinction coefficients (ε) as well as high fluorescence quantum yields (Φf), is still a demand for optical imaging. We now present a synthetic route for the preparation of a new group of glycerol-substituted cyanine dyes from dendronized indole precursors that have been functionalized as N-hydroxysuccinimide (NHS) esters. High Φf values of up to 0.15 and extinction coefficients of up to 189 000 L mol(-1) cm(-1) were obtained for the pure dyes. Furthermore, conjugates of the new dendronized dyes with the antibody cetuximab (ctx) that were directed against the epidermal growth factor receptor (EGFR) of tumor cells could be prepared with dye to protein ratios between 0.3 and 2.2 to assess their potential as imaging probes. For the first time, ctx conjugates could be achieved without showing a decrease in Φf and with an increasing labeling degree that exceeded the value of the pure dye even at a labeling degree above 2. The incorporation of hydrophilically and sterically demanding dendrimers into cyanines prevented dimer formation after covalent conjugation to the antibody. The binding functionality of the resulting ctx conjugates to the EGFR was successfully demonstrated by cell microscopy studies using EGFR expressing cell lines. In summary, the combination of hydrophilic glycerol dendrons with reactive dye labels has been established for the first time and is a promising approach toward more powerful fluorescent labels with less dimerization.

In mammalian skin, stem cell factor (SCF) regulates the proliferation and maturation of mast cells and melanocytes, which are thought to be the only cutaneous cells that express the Kit-tyrosine kinase receptor (Kit) and respond to epithelial and mesenchymal-derived SCF. We previously had noted, however, the presence of Kit+ cells in murine hair follicles, in an introepithelial tissue compartment devoid of melanocytes and mast cells. Here we have identified the nature of this Kit+ population of cells in hair follicles of C57BL/6 mice. Anagen hair follicles showed strong Kit immunoreactivity not only in the pigmentary unit above the follicular dermal papilla but also in a much more proximally located, homogenous group of nondendritic, nonmelanized cells. By immunohistochemistry (desmoplakin+/Trp-1-) and electron microscopy (presence of tonofilaments, desmosomes, lack of melanosomes), these Kit+ cells were shown to be hair matrix keratinocytes and were also found in melanocyte-deficient hair follicles (Kit(Sl)/Kit(Sl-d) mice, Kit-neutralizing antibody-treated C57BL/6 mice). Expression of Kit and SCF was strongly hair-cycle-dependent, suggesting a functional role of epithelial Kit expression in hair growth control. This was supported by the observation that mice unable to respond to SCF stimulation (Kit(W)/Kit(W-v)) showed a significant retardation of anagen development compared to their wild-type littermates. The expression of Kit in the most rapidly proliferating compartment of the hair follicle epithelium suggests intriguing, as yet unexplored new functions of Kit signaling in epithelial cell biology.

Background: Receptors for vasoactive intestinal polypeptide (VIP) have recently been suggested to play a key role in immunomodulation with genetically modified mice. However, it is not known whether changes in receptor gene regulation are involved in the pathogenesis of human immune disorders. Objective: We studied the expression of VPAC 2 in acute lesions of the human immune disease atopic dermatitis. Methods: By using nonradioactive in situ hybridization, quantitative immunohistochemistry, RT-PCR, and gene array studies, the expression status of VPAC 2 was assessed in atopic dermatitis and control tissues and in the human mast cell line HMC-1. Results: In situ hybridization and immunohistochemistry demonstrated VPAC 2 mRNA and protein expression in human mast cells surrounded by VIP positive nerve fibers. Gene array experiments and RT-PCR studies showed high levels of VPAC 2 mRNA expression in mast cells that were increased compared to other receptors such as VPAC 1 or VIP in the human mast cell line HMC-1. Stimulation of HMC-1 cells led to a downregulation of VPAC 2 . Similarly, quantitative immunohistochemistry for VPAC 2 in acute atopic dermatitis lesions showed a significantly decreased VPAC 2 immunoreactivity in mast cells. Conclusion: The downregulation of VPAC 2 in human mast cells in acute lesions of atopic dermatitis suggests a role of this G-protein;coupled receptor in the pathophysiology of the disease. (J Allergy Clin Immunol 2003;111:1099-105.)

In this study, dendritic polyglycerol sulfate (dPGS) is evaluated as a delivery platform for the anticancer, tubulin-binding drug paclitaxel (PTX). The conjugation of PTX to dPGS is conducted via a labile ester linkage. A non-sulfated dendritic polyglycerol (dPG) is used as a control, and the labeling with an indocarbocyanine dye (ICC) renders multifunctional conjugates that can be monitored by fluorescence microscopy. The conjugates are characterized by (1)H NMR, UV-vis measurements, and RP-HPLC. In vitro cytotoxicity of PTX and dendritic conjugates is evaluated using A549 and A431 cell lines, showing a reduced cytotoxic efficacy of the conjugates compared to PTX. The study of uptake kinetics reveals a linear, non saturable uptake in tumor cells for dPGS-PTX-ICC, while dPG-PTX-ICC is hardly taken up. Despite the marginal uptake of dPG-PTX-ICC, it prompts tubulin polymerization to a comparable extent as PTX. These observations suggest a fast ester hydrolysis and premature drug release, as confirmed by HPLC measurements in the presence of plasma enzymes.

Inflammatory processes driven by cytokines play a crucial role during osteoarthritis (OA) progression. Dendritic polyglycerol sulfate (dPGS) was analyzed in vitro for its effects on articular chondrocytes, cartilage and cytokines involved in the OA process.The metabolic activity of cultured human articular chondrocytes stimulated for 24 h with dPGS (10(-3)-10(-6) mol/L) was monitored using AlamarBlue(®) assay. The dPGS uptake was studied using fluorescence labeled nanoparticles. Further, chondrocytes were either treated with 10(-6) M dPGS, TNFα (10 ng/mL) alone or with a combination of both. The influence on extracellular matrix components, pro- and anti-inflammatory cytokines, matrix metalloproteinase (MMP)1 and the anaphylatoxin receptor C3aR was analyzed by RTD-PCR, flow cytometry and ELISA.Even at higher dosages (10(-3) mol/L), dPGS did not influence chondrocytes viability. Uptake of dPGS was successfully monitored in human articular chondrocytes and synovial fibroblasts, penetration into cartilage chips was up to ~50 µm. Cellular treatment with dPGS had no effect on synthesis of pro-inflammatory cytokines TNFα and IL-6, but expression of the anti-inflammatory IL-10 was upregulated. Cotreatment with TNFα and dPGS reduced the TNFα level, while IL-1β, IL-6 and IL-10 expression did not change. Collagen type II gene expression was significantly reduced after preincubating cells with dPGS, but remained unaffected at the protein level.Results indicate that dPGS could play a role in regulation of cytokines associated with the inflammatory aspect of OA progression.

Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) doxorubicin (Dox) linked via a pH-sensitive hydrazone to (c) a tri-functional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Dox. The drug fluorescence is quenched via intramolecular FRET until the pH-sensitive hydrazone bond between the TMP and Dox is cleaved at acidic pH. By measuring its fluorescence, we characterized the TMP cleavage kinetics at different pH values in vitro. The intracellular release of Dox from the carrier was monitored in real time in intact cancer cells, giving more insight into the mode of action of a polymer drug conjugate.

Synthesis of Dye-Labeled Polyanions In previous studies native and demineralized ovine bone was used to analyze the affinity of polyanions derived from dendritic polyglycerol (dPG) towa Selective Targeting of Bone and Cartilage with Multivalent Dendritic Polyanions Sabine Reimann1, Dominic Groger1, Ngee Han Lim2, Kai Licha3, Pia Welker Peter Fratzl5, Rainer Haag 1Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Takustr. 3, 14195 Berlin, of Medicine, Imperial College London, 1 Aspenlea Rd, London W6 8LH, UK 3Zentralinstitut Universitatsmedizin Berlin, CBF, Hindenburgdamm 30, 12203 Berlin, Germany 4Charite-Universitatsmedizin und Wiederherstellungschirurgie Garystr. 5, D-14195, Berlin, Germany 5Max Planck Institute Potsdam, Germany.

Polyglycerol based nanogels (nPG) can function as cellular delivery systems. These nPGs are synthesized with different amine densities (nPG amines) by acid-catalyzed epoxide-opening polymerization using a mini-emulsion approach and surface modification. All the synthesized nanogels are characterized by NMR, dynamic light scattering, and ζ-potential, showing slightly positive surface charge and a homogeneous size of ≈100 nm. The use of these systems for delivery applications is demonstrated with regard to polyplex formation, cytotoxicity, and cellular uptake studies. It is depicted that the CE50 value of the high loaded nPG amines is eight times higher than the low loaded ones. The influence of the amine loading percentage on the nanogel and the effects of polyvalency in these architecture is discussed.

Several groups have previously reported that rodent or human leukemic mast cells produce inflammatory cytokines such as TNF-alpha and IL-8 as well as the pro-allergic cytokines IL-4, IL-5 and IL-13. Comparatively little is known, however, regarding the ability of normal human skin mast cells to secrete these factors following either IgE-dependent or IgE-independent modes of activation. We therefore investigated whether normal human skin mast cells produce these cytokines following stimulation by a variety of secretagogues. Enriched isolated skin mast cells released both TNF-alpha and IL-8 following activation with either anti-IgE, SCF, substance P, compound 48/80 or A23187. This release was dose- and time-dependent, with maximal levels being reached within 4 h of stimulation involving, in part, the secretion of preformed stores of both cytokines. In accordance with this, using lysates of highly purified (>90%) skin mast cells, we could demonstrate that both TNF-alpha and IL-8 mRNA and protein were present in both unstimulated as well as stimulated mast cells. In stark contrast to these results, no significant levels of either IL-4, IL-5 or IL-13 were detected, regardless of the secretagogue used or the period of stimulation. These results show that human skin mast cells are capable of rapidly secreting pro-inflammatory cytokines like TNF-alpha and IL-8 following IgE-dependent activation and stimulation by the neuropeptide substance P, SCF and the basic polypeptide analogue compound 48/80. In contrast to other types of human mast cells however, human skin mast cells were incapable of secreting IL-4, IL-5 or IL-13 in these settings.

In order to explore possible mechanisms involved in the previously documented turnover of mast cell subpopulations in human cutaneous scars, we have examined selected factors known to stimulate and/or modulate mast cell hyperplasia (SCF, NGF, TGFbeta1, GM-CSF) and their receptors in human cutaneous scar tissue. On immunohistochemistry, numbers of SCF- and TGFbeta1-positive cells were significantly increased in the epidermis and throughout the dermis in scars (n = 27) of varying ages (4-369 d old), compared with normal skin (n = 12). Furthermore, TRbetaRI, II, and the NGF-p75 receptors were significantly increased in the epidermis, TRbetaRI and NGF-TrkA throughout the dermis, and TRbetaRII, NGF-p75, and GM-CSFR only in the mid- and lower dermis of scars. NGF and GM-CSF expression was in contrast scarce and weak, with no differences between normal skin and scars. In tissue extracts, mRNA levels of SCF, TGFbeta1, TRbetaI and II, and both NGF-receptors, but not GM-CSFR, were significantly increased as well. TRbetaI and II were identified in up to 90% and 83%, respectively, of isolated normal skin mast cells on flow cytometry, and GM-CSFR and NGFR-p75 were identified on 70% and 73%, respectively, of avidin-positive normal mast cells on double immunofluorescence microscopy. As described before for the SCF receptor KIT, GM-CSFR and NGFR-p75 were partly or entirely downregulated on avidin-positive mast cells in scars. The marked upregulation of TGFbeta1, its type I and II receptors, and SCF suggest that these factors play a major role in the orchestration of mast cell increase in human cutaneous scars whereas the role of NGF and GM-CSF is less clear, despite the significant upregulation of their receptors.

SUMMARY Although the intercellular adhesion molecule-1 (ICAM-1) is recognized for its pivotal role in inflammation and immune responses, its role in developmental systems, such as the cyclic growth (anagen) and regression (catagen) of the hair follicle, remains to be explored. Here we demonstrate that ICAM-1 expression in murine skin is even more widespread and more developmentally regulated than was previously believed. In addition to endothelial cells, selected epidermal and follicular keratinocyte subpopulations, as well as interfollicular fibroblasts, express ICAM-1. Murine hair follicles express ICAM-1 only late during morphogenesis. Thereafter, morphologically identical follicles markedly differ in their ICAM-1 expression patterns, which become strikingly hair cycle-dependent in both intra- and extrafollicular skin compartments. Minimal ICAM-1 and leukocyte function-associated (LFA-1) protein and mRNA expression is observed during early anagen and maximal expression during late anagen and catagen. Keratinocytes of the distal outer root sheath, fibroblasts of the perifollicular connective tissue sheath, and perifollicular blood vessels exhibit maximal ICAM-1 immunoreactivity during catagen, which corresponds to changes of LFA-1 expression on perifollicular macrophages. Finally, ICAM-1-deficient mice display significant catagen acceleration compared to wild-type controls. Therefore, ICAM-1 upregulation is not limited to pathological situations but is also important for skin and hair follicle remodeling. Collectively, this suggests a new and apparently nonimmunological function for ICAM-1-related signaling in cutaneous biology. (J Histochem Cytochem 48:557‐568, 2000)

H1-type antihistamines have recently been reported to inhibit cytokine secretion from human and murine mast cells and basophils. In order to confirm and expand these studies, we have compared several H1-blockers and the H2-blocker ranitidine for their effect on TNF-alpha, IL-3, 6, 8 and GM-CSF release from human leukemic mast (HMC-1) and basophilic (KU812) cells, compared to dexamethasone. Cells were stimulated for 24 h with phorbol myristate acetate (25 ng/ml) and calcium ionophore A 23187 (2.5x10(-7) M) alone or with the drugs added at 10(-4) to 10(-15) M, and production of cytokines was measured by ELISA. All antihistamines caused a dose-dependent inhibition of TNF-alpha release from HMC-1 cells, with maximal effects at 10(-12) M for azelastine, 10(-9) M for loratadine and cetirizine, and 10(-8) M for ranitidine. The inhibitory potency of H1-blockers on cytokines from HMC-1 cells was TNF-alpha >IL-8> or =IL-6> or =IL-3, with no significant effects on GM-CSF. In KU812 cells which failed to secrete TNF-alpha and GM-CSF, the sequence was IL-6 >IL-8 after preincubation. Dexamethasone inhibited all cytokines, but ranitidine only TNF-alpha and IL-3. Antihistamines had no effect on calcium flux in resting or stimulated cells. At the mRNA level, inhibition was only seen with KU812 cells and IL-8 in the presence of azelastine at 10-(10) M. These data show thus distinct inhibitory patterns for different antihistamines during cytokine production from human mast cells and basophils which may contribute to the anti-inflammatory effects of these drugs during treatment of allergic diseases.

Nerve growth factor-β (NGF) is known as a growth factor for human basophils and murine mast cells and has recently been shown to also up-regulate mast cell characteristics in human leukaemic mast cells. We have examined here the effect of NGF on the differentiation of normal human mast cells from cord blood progenitors during culture with stem cell factor (SCF), NGF alone or in combination, or fibroblast supernatants. All these supplements induced mast cell immunoreactivity against tryptase, c-Kit and FceRIα, but none of the cells reacted against the basophil specific antibody 2D7 before or during culture. Intracellular tryptase activity increased as well, with maximal levels on combined culture with SCF and NGF. On reverse transcription–polymerase chain reaction (RT–PCR), cells lacked tryptase and chymase and expressed low levels of FceRI and c-Kit mRNA prior to culture, with marked up-regulation of FceRI and c-Kit, and with de novo expression of mast-cell specific α- and β-tryptase by week 3, and of chymase by week 5. Only the TrkA and not the p75 NGF receptor was detected at m-RNA and protein level, and only the TrkA NGF receptor was up-regulated during NGF-driven culture. These findings show therefore that, like SCF, NGF is another growth factor that can induce and regulate human mast-cell development and differentiation.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and their receptors, GDNF family receptor α-1 (GFRα-1) and GDNF family receptor α-2 (GFRα-2), are critically important for kidney and nervous system development. However, their role in skin biology, specifically in hair growth control, is as yet unknown. We have studied expression and function of GDNF, neurturin, GFRα-1, and GFRα-2 in murine skin during the cyclic transformation of the hair follicle (HF) from its resting state (telogen) to active growth (anagen) and then through regression (catagen) back to telogen. GDNF protein and GFRα-1 messenger RNA are prominently expressed in telogen skin, which lacks NTN and GFRα-2 transcripts. Early anagen development is accompanied by a significant decline in the skin content of GDNF protein and GFRα-1 transcripts. During the anagen-catagen transition, GDNF, GFRα-1, NTN, and GFRα-2 transcripts reach maximal levels. Compared with wild-type controls, GFRα-1 (+/−) and GFRα-2 (−/−) knockout mice show a significantly accelerated catagen development. Furthermore, GDNF or NTN administration significantly retards HF regression in organ-cultured mouse skin. This suggests important, previously unrecognized roles for GDNF/GFRα-1 and NTN/GFRα-2 signaling in skin biology, specifically in the control of apoptosis-driven HF involution, and raises the possibility that GFRα-1/GFRα-2 agonists/antagonists might become exploitable for the treatment of hair growth disorders that are related to abnormalities in catagen development.

HGF/SF and its receptor (Met) are principal mediators of mesenchymal-epithelial interactions in several different systems and have recently been implicated in the control of hair follicle (HF) growth. We have studied their expression patterns during HF morphogenesis and cycling in C57BL/6 mice, whereas functional hair growth effects of HGF/SF were assessed in vivo by analysis of transgenic mice and in skin organ culture. In normal mouse skin, follicular expression of HGF/SF and Met was strikingly localized: HGF/SF was found only in the HF mesenchyme (dermal papilla fibroblasts) and Met in the neighboring hair bulb keratinocytes. Both HGF/SF and Met expression peaked during the initial phases of HF morphogenesis, the stage of active hair growth (early and mid anagen), and during the apoptosis-driven HF regression (catagen). Met+ cells in the regressing epithelial strand appeared to be protected from undergoing apoptosis. Compared to wild-type controls, transgenic mice overexpressing HGF/SF under the control of the MT-1 promoter had twice as many developing HF and displayed accelerated HF development on postnatal day 3. They also showed significant catagen retardation on P17. In organ culture and in vivo, HGF/SF i.c. resulted in a significant catagen retardation. These results demonstrate an important role of HGF/SF and Met in murine hair growth control and suggest that Met-mediated signaling might be exploited for therapeutic manipulation of human hair growth disorders.-Lindner, G., Menrad, A., Gherardi, E., Merlino, G., Welker, P., Handjiski, B., Roloff, B., Paus, R. Involvement of hepatocyte growth factor/scatter factor and Met receptor signaling in hair follicle morphogenesis and cycling.

In order to explore a possible involvement of mast cells during human wound healing, we studied sections from scars (4-369-d-old) (N = 20) and normal skin (N = 10) for mast-cell-specific tryptase and chymase by enzyme histochemistry, for the stem cell factor receptor c-Kit and the melanosomal marker TA99 by immunohistochemistry, and for simultaneous c-Kit expression and avidin fluorescence by double staining. Enzyme activities and mRNA expression were also studied in tissue extracts. Chymase-reactive mast cell numbers as well as chymase activity and mRNA expression were reduced in all scars, whereas overall numbers of tryptase-reactive cells did not differ from normal skin, although tryptase activity and mRNA expression were increased in scar extracts. In contrast, numbers of c-Kit positive cells were significantly increased in old scars, and in the mid and lower dermis of all scars. A marked reduction of c-Kit reactivity was noted, however, in avidin-positive dermal mast cells and in epidermal basal cells, despite unchanged numbers of melanosome-positive cells, with an associated overall decrease of c-Kit mRNA in scar extracts. These data thus show that numbers of resident mast cells are very low in human cutaneous scars, suggesting massive mediator release from these cells into fresh wounds. Downregulation of stem cell factor receptors may also prevent these cells from increasing in number even in old scars. Instead, scar tissue is populated by a mast cell subpopulation that is chymase-, avidin-, tryptase +, c-Kit +, reflecting most probably an increased immigration and/or proliferation of immature mast cells and their precursors.

Stem cell factor (SCF), characterized as mast cell growth factor, is known to be produced by fibroblasts, keratinocytes and endothelial cells. Two different splice variants encode for either a soluble (SCF-1) or a membrane-bound (SCF-2) form. In order to explore whether mast cells themselves can produce SCF, we examined cultured cord blood (CBMC) and peripheral-blood-derived mast cells (PBMC), mast/basophil cell lines (HMC-1 and KU-812), and skin mast cells for SCF expression. On immunocytochemistry, cytoplasmatic SCF-reactivity was observed in HMC-1 cells, with additional cell membrane staining in KU-812, skin and cultured mast cells. Low amounts of SCF could be detected by ELISA in lysates of isolated and unstimulated mast cells and in supernatants of skin cells stimulated with anti-IgE or Ca-ionophore A23187. SCF mRNA was detected in all cells, although marked quantitative differences were observed among the various cell types. SCF-2 mRNA expression was low in HMC-1 cells while it was marked in skin mast cells, KU-812 cells, CBMC and PBMC. A time-dependent, increasing induction of both SCF forms was seen in CBMC and PBMC during culture. After stimulation with A23187 and phorbol myristate acetate, an up-regulation of SCF mRNA was noted in HMC-1 and KU-812 cells, without changes in the relationship of the two splice variants. The differential expression of SCF-specific mRNA splice variants in immature and mature human mast cells and the secretion of this molecule by these cells may play a role in autocrine stimulation, maintenance of survival and the differentiation of tissue mast cells.

Mastocytosis represents a mast cell proliferative disease that generally runs a benign clinical course, with spontaneous remissions mostly by puberty in childhood-onset disease, although rare forms, particularly in adult-onset disease, can be associated with (pre)malignant hematologic disorders and very rarely present as mast cell leukemia or malignant mastocytosis. Reasons for this divergent clinical behavior of childhood- versus adult-onset disease are unknown. Recently, two activating mutations in the intracellular domain of the proto-oncogene c-kit, which encodes a tyrosine kinase receptor for the mast cell growth factor stem cell factor, have been detected in the human leukemic mast cell line HMC-1. We have therefore studied lesional skin biopsies from patients with adult- and childhood-onset indolent mastocytosis for the presence of these codon 560 and 816 mutations. C-kit coding DNA sequences were amplified and analyzed by mutation-specific restriction analyses, and mutated polymerase chain reaction products were additionally cloned and sequenced. The codon 816 mutation was found in all six samples from adult patients, but not in any of the 11 specimens from children. In addition, the codon 560 mutation could be demonstrated for the first time in indolent mastocytosis, namely in two of four specimens from adult patients, but not in those from two children. These data thus provide a possible explanation for the divergent clinical behavior of adult- versus childhood-onset indolent mastocytosis, with the first being associated with an activating mutation, possibly as part of a neoplastic process, and the latter representing most likely a reactive process of an as yet unknown pathogenesis.

Nervous system and hair follicle epithelium share a common ectodermal origin, and some neurotrophins (NTs) can modulate keratinocyte proliferation and apoptosis. Therefore, it is reasonable to ask whether NTs are also involved in hair growth control. Here, we show that the expression of NT-3 and its high-affinity receptor, tyrosine kinase C, in the skin of C57BL/6 mice is strikingly hair cycle-dependent, with maximal transcript and protein expression seen during spontaneous hair follicle regression (catagen). During catagen, NT-3 and tyrosine kinase C are co-expressed by terminal deoxynucleotidyl transferase-mediated in situ nick end labeling-positive keratinocytes in the club hair and secondary germ. NT-3-overexpressing transgenic mice show precocious catagen development during the postnatal initiation of hair follicle cycling, whereas heterozygous NT-3 knockout (+/−) mice display a significant catagen retardation. Finally, NT-3 stimulates catagen development in organ culture of normal C57BL/6 mouse skin. These observations suggest that the hair follicle is both a source and target of NT-3 and that NT-3/tyrosine kinase C signaling is functionally important in the control of hair follicle regression. Therefore, tyrosine kinase C agonists and antagonists deserve systematic exploration for the management of hair growth disorders that are related to premature (alopecia/effluvium) or retarded catagen (hirsutism/hypertrichosis).

In order to explore the potential existence of human mast cell growth factors other than stem cell factor (SCF), we have compared SCF to L-cell fibroblast supernatants (LCS) during in vitro mast cell differentiation, using human leukaemic mast cells (HMC-1 cells) which contain a gain-of-function mutated SCF receptor (c-Kit) as model. At baseline, cells exhibited an immature phenotype, with

Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.

A set of six hydrophobically derivatized polymers based on polyglycerol sulfates have been investigated to determine the influence of scaffold architecture on the encapsulation properties of hydrophobic guests. Each of three block and statistical copolymers has been synthesized with phenyl, naphthyl, and biphenyl substituents in a one-pot procedure. The copolymers have been functionalized with sulfate groups in order to introduce an electrostatically repulsive surface that can stabilize the aggregated carriers. In addition, sulfates provide a highly active targeting moiety for inflammation and cellular uptake. UV measurements show a supramolecular encapsulation of the investigated guest molecules in the low μM range. The transport studies with pyrene and an indocarbocyanine dye further indicated a core–shell-type architecture which provides a distinct amphiphilicity as required for supramolecular guest complexation. The combination of a host functionality with an active sulfate targeting moiety has been used to investigate the structure related cellular transport properties.

Mast cells are not only the primary effector cells of immediate type immune reactions, but they have recently also been considered to contribute to the induction of an immune response. Data on the ability of the cells to express major histocompatibility complex (MHC) antigens and the mechanisms involved are however controversial or unclear. We have therefore studied the expression of MHCI and the induction of MHCII molecules on leukemic HMC-1 mast cells by immunohistochemistry. Cells were incubated for up to 72 h in the presence of IFN gamma, TNF alpha and IL-4, and immunohistochemical staining was done with monoclonal antibodies with specificity for HLA class I heavy chain, HLA-DQ (Tü22), HLA-DR (Tü36) and HLA-DQ, -R and -P (Tü35). All unstimulated mast cells expressed MHC class I, but almost no class II antigens. Incubation with IFN gamma caused a rapid, dose-dependent induction of MHC class II molecules, with Tü35 staining maximally one third of the cells within 24 h at the highest dose tested (100 IU/ml), with decline on extended culture. TNF alpha (2 ng/ml) was less effective but caused more persistent induction with time. IL-4 (200 ng/ml) had hardly any effects at all. Staining with Tü22 and Tü36 was always lower than with Tü35, and additive or even synergistic results were obtained when cells were stimulated with a combination of IFN gamma and TNF alpha. These findings support the concept that mast cells can facultatively participate in immune recognition processes depending on the type of pathological conditions in their microenvironment which allow expression of MHC class II molecules.