Role of the novel mTOR inhibitor RAD001 (everolimus) in anaplastic thyroid cancer.

Activation of the phosphatidylinositol-3-kinase (PI3K) signaling cascade is increasingly recognized as a common feature of thyroid follicular neoplasms. Among the PI3K downstream effectors, the main kinase, directly responsible for the increased cell growth and proliferation, is called mammalian target of rapamycin (mTOR). This central kinase might be directly inhibited via rapamycin and its derivatives. The aim of the present study was to examine whether RAD001 (everolimus) can selectively suppress the proliferation of different anaplastic thyroid cancer (ATC) cells. Five different human ATC cell lines were exposed to different concentrations of RAD001. Importantly, we found a dose-dependent growth inhibition in two ATC cell lines at concentrations of 43.5 and 94.5 nM although not as intensive as within the RAD001 responding K562cell line. The other cell lines revealed a GI (50) between 168 to 234 nM. In parallel, quantitative PCR of PCNA displayed a reduced expression of PCNA within the responding cell lines, respectively. In summary, we found a good responding effect in a part of ATC cell lines, which may have a clinical impact.

Chromogranin a as potential tumour antigen in pheochromocytoma.

Chromogranin A (CgA) is a dense core vesicle-associated protein and, therefore, represents a specific marker of neuroendocrine cells and tumours including pheochromocytomas. CgA has already been investigated for its immunogenic properties. Importantly, in vitro studies demonstrated the presence of naturally occurring CgA-specific cytotoxic T cells in patients with neuroendocrine cancers. Therefore, it is worthwhile to investigate the potential role of CgA as tumour antigen in pheochromocytoma. Depending on the results of these ongoing in vitro and in vivo studies, a clinical application may arise in the near future.

Cellular therapies in endocrine diseases.

Currently, no curative therapies are available for many malignant diseases and for autoimmune disorders. Over the past two decades, however, substantial progress has been made in our understanding of immunity and immune tolerance. Numerous of immune mechanisms were identified responsible for breaking the state of tolerance and for inducing specific cytotoxic immunity. In parallel, much knowledge has been gained on how immune tolerance can be re-induced in autoimmune diseases. Here, we would like to add value to the current advances in cellular therapy for treating endocrine cancer as well as for inducing immune tolerance in autoimmune diseases. In addition, new insights into stem cell research in autoimmune diseases are presented and future perspectives are given.

Dendritic cell subtypes and in vitro generation of dendritic cells.

Dendritic cells (DCs) are highly potent antigen-presenting cells crucial for the innate and adaptive immune response and for maintaining immune tolerance towards self-antigens. Although they share many common features, multiple DC subtypes with different immune functions have been identified. Originally, DCs were considered to be cells with purely myeloid origin. Recent studies have now demonstrated that DCs can also develop from lymphatic progenitors. Various cytokines and transcription factors are known to be responsible for the development of DC subpopulation. Depending on the subpopulation and the maturation state of these cells, they are either able to induce a broad cytotoxic immune response, and therefore represent a promising tool for anticancer vaccination therapies in humans or induce immune tolerance and are important within the context of autoimmunity. This review will focus on recent advances on the identification of different DC subpopulations including phenotypical and functional differences and on recent developments on protocols for IN VITRO generation of myeloid-derived DCs.

Dendritic cell vaccination induces tumor epitope-specific Th1 immune response in medullary thyroid carcinoma.

The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC) and their resistance to classical therapies make it a prime candidate for adoptive immunotherapy. Highly potent antigen-presenting cells, namely dendritic cells (DCs), may serve as an interesting tool for anticancer vaccination. Here we report on the IN VITRO findings of a vaccination trial in five MTC patients, who were treated with a new DC generation protocol consisting of granulocyte-macrophage colony-stimulating factor and interferon-alpha (IFN-DCs). These cells were pulsed with tumor-specific calcitonin and administered twice. In two patients who responded to therapy we found a large increase (in mean 2.9+/-1.9%) of antigen-specific IFN-gamma-secreting CD4+ cells as well as an increase of granzyme B positive CD8+ cells (mean 2.2+/-0.2%) in the peripheral blood. In parallel, a decrease of CD4+/CD25+/FoxP3+ regulatory T cells was seen. Importantly, IN VITRO stimulation of PBMC with 10 different 15mer calcitonin peptides resulted in the identification of two HLA class II epitope regions within the central part of full-length calcitonin. These data were in accordance with the results drawn from the computer-based algorithm epitope prediction software SYFPEITHI. Measurement of different pro- and anti-angiogenic factors did not allow for a distinct outcome of prediction of the treated patients. In summary, we have demonstrated that immunization with IFN-DCs leads to a tumor epitope-specific immune response in MTC patients and may, therefore, represent a promising tool for future vaccination trials.

Characterization of monocyte-derived IFNalpha-generated dendritic cells.

The antitumor effects of IFNalpha is mainly mediated by the activation of cytotoxic T lymphocytes (CTLs), the activation of natural killer (NK) cells, and the generation of highly potent antigen-presenting dendritic cells (IFN-DCs). Recently, we demonstrated that these cells partially express the NK cell marker CD56 and reveal a direct cytotoxic immunity towards tumor cells. The aim of the present study was to explore these cells in more detail with respect to their phenotypical and functional characteristics. Flowcytometric analyses revealed that a 5-day incubation time of CD14+ monocytes with IFNalpha results in a steady increase of CD56 surface expression of these cells from 25% (+/-2%) on day 1 up to 68% (+/-11%) on day 5. Interestingly, additional culturing of negatively selected CD56- IFN-DCs also resulted in a partial CD56 surface expression. By comparing both cell types in more detail we found a significant decrease of CD14 expression on CD56+ IFN-DCs (66+/-6%) compared to CD56- IFN-DCs (76+/-6%). On the basis of functional tests, CD56+ IFN-DCs revealed a slightly increased phagocytosis capacity compared to CD56- IFN-DCs as only 82% of CD56- IFN-DCs showed a positive intracytoplasmatic signal after 60 minutes coculturing with FITC-labeled albumin, whereas 91% of CD56+ IFN-DCs were positive. Moreover, CD56+ IFN-DCs revealed a stronger T cell stimulation capacity compared to CD56- IFN-DCs. These results together with our previously described data suggest that CD56+ IFN-DCs and CD56- IFN-DCs may represent one identical cell population with different maturation status rather than two separate cell entities. Because of their high stimulating capacity and their direct cytolytic effects these cells represent a new promising tool for cellular anticancer therapy.

Indirect lymph node lymphangiography using an iodine-based contrast medium and projection radiography following submucosal injection in a rabbit model.

BACKGROUND: This study investigated the possibility of local lymph node detection and lymphatic mapping following submucosal injection of an iodine-based contrast medium. METHODS AND MATERIALS: We established a contrast medium (oil/water emulsion on iodine basis) with a particle size of mainly 1.7+/-0.1 micro m. Ten rabbits received rectal submucosal injections of the contrast medium and underwent repeated projection radiography. RESULTS: Passage of the contrast medium into lymphatic vessels and storage in lymph nodes was seen in all ten animals. The best contrast was achieved within 24 and 48 h after injection. Lymph nodes were still seen in eight cases with the final radiograph on day 14. There were no clinical side effects observed. Injection sites showed mild signs of inflammation in histological examinations. Pathological signs were not detectable in lymph nodes containing the contrast media. CONCLUSIONS: This method appears useful when investigating local lymph nodes following submucosal injection due to its passage into lymphatic vessels and storage in lymph nodes.

[Fracture of the acromion. Diagnosis--treatment strategy--outcome]. / Die Akromionfraktur. Diagnostik--Behandlungsstrategie--Ergebnis.

The case of an 22-year-old man is presented, who sustained a dislocated fracture of the left acromion process and a not dislocated fracture of the left scapular body with a large subcutaneous décollement as well as a dammage of nervus axillaris occurring during a traffic accident. After resuming diagnostics by means of CT, a tension banding of the ventral part of the acromion and a plate osteosynthesis of the dorsal part was performed. 7 weeks after injury neurolysis of nervus axillaris has been done. 4 months after accident the patient shows a satisfying functional result in the Constant score. Diagnostic, treatment and functional results after operative treatment of dislocated fractures of the acromion are shown and discussed.

Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding.

Human bone sialoprotein (BSP) comprises 15% of the total noncollagenous proteins in bone and is thought to be involved in bone mineralization and remodeling. Recent data suggest a role for BSP in breast cancer and the development of bone metastases. We have produced full-length recombinant BSP in a human cell line and purified the protein from human bone retaining the native structure with proper folding and post-translational modifications. Mass spectrometry of bone-derived BSP revealed an average mass of 49 kDa and for recombinant BSP 57 kDa. The post-translational modifications contribute 30-40%. Carbohydrate analysis revealed 10 different complex-type N-glycans on both proteins and eight different O-glycans on recombinant BSP, four of those were found on bone-derived BSP. We could identify eight threonines modified by O-glycans, leaving the C terminus of the protein free of glycans. The recombinant protein showed similar secondary structures as bone-derived BSP. BSP was visualized in electron microscopy as a globule linked to a thread-like structure. The affinity for hydroxyapatite was higher for bone-derived BSP than for recombinant BSP. Cell adhesion assays showed that the binding of BSP to cells can be reversibly diminished by denaturation.

Bicarbonate-regulated soluble adenylyl cyclase.

Soluble adenylyl cyclase (sAC) represents a novel form of mammalian adenylyl cyclase structurally, molecularly, and biochemically distinct from the G protein-regulated, transmembrane adenylyl cyclases (tmACs). sAC possesses no transmembrane domains and is insensitive to classic modulators of tmACs, such as heterotrimeric G proteins and P site ligands. Thus, sAC defines an independently regulated cAMP signaling system within mammalian cells. sAC is directly stimulated by bicarbonate ion both in vivo in heterologously expressing cells and in vitro using purified protein. sAC appears to be the predominant form of adenylyl cyclase (AC) in mammalian sperm, and its direct activation by bicarbonate provides a mechanism for generating the cAMP required to complete the bicarbonate-induced processes necessary for fertilization, including hyperactivated motility, capacitation, and the acrosome reaction. Immunolocalization studies reveal sAC is also abundantly expressed in other tissues which respond to bicarbonate or carbon dioxide levels suggesting it may function as a general bicarbonate/CO(2) sensor throughout the body.

Olfactory adaptation in Drosophila larvae.

To accommodate both high sensitivity as well as the ability to respond to a broad range of stimulus concentrations, an organism must possess some means of modulating the gain of its sensory systems. This phenomenon is known as adaptation. Here, we demonstrate that Drosophila larvae can adapt to three odorants in a behavioral paradigm. Larval olfactory adaptation is concentration- and dose-dependent. Olfactory and visual adaptation in Drosophila melanogaster adults is dependent on the transient receptor potential (trp) calcium channel. Recovery from olfactory adaptation, which is TRP-dependent in adults, is shown to be unaffected by a loss-of-function trp mutation in larvae. Moreover, the TRP gene product is not expressed in the larval olfactory organs. These observations suggest a role for trp in mediating sensory function that is conserved between sensory modalities in adults but is not conserved between developmental stages.

[Immobilizing muscle weakness accentuated in leg and proximal muscles]. / Immobilisierende bein- und proximalbetonte Muskelschwäche.

A 54 year old waiter was referred to the hospital because of proximal muscle weakness, most pronounced in his legs, which progressed to an inability to stand or walk within weeks. Myopathy was diagnosed based on the muscle biopsy findings and myositis was ruled out by laboratory and biopsy results. Further investigations led us to exclude an endocrine cause, hypovitaminosis D, infectious myopathy or a paraneoplastic syndrome. Heteroanamnesis revealed severe alcoholism, lasting for more than 30 years. The presumed alcohol induced hepatopathy was confirmed by liver biopsy. There were no signs of an acute alcoholic myopathy, as the weakness had developed rather insidiously, there was no elevation of the CK serum level nor myoglobinuria and a type 2 fibre atrophy was found by muscle biopsy. As expected the weakness improved under abstention. Thus the final diagnosis of a chronic alcohol induced myopathy was established.

Chronic myelogenous leukemia: effect of interferon-alpha treatment on phagocytic activity and capacity of circulating neutrophils.

This study focuses on the effect of interferon (IFN)-alpha on phagocytosis of FITC-labeled Escherichia coli by polymorphonuclear neutrophils (PMNs) in chronic myelogenous leukemia (CML). The phagocytic activity and capacity of PMNs from IFN-alpha treated patients (n = 17), untreated CML patients (n = 9) and from healthy donors (n = 20) were compared using flow cytometry. Both parameters of PMN phagocytosis were reduced in untreated CML and in IFN-alpha treated CML with Ph1 chromosome persistence but normal in IFN-alpha treated CML with Ph1 conversion. Thus, the phagocytic performance of PMNs in patients with chronic phase CML is significantly improved after successful treatment with IFN-alpha.

YAC contigs of the Rab1 and wobbler (wr) spinal muscular atrophy gene region on proximal mouse chromosome 11 and of the homologous region on human chromosome 2p.

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glnsps1, an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping. Rab1 and Glns-ps1 were found to be only 200 kb apart. A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found between these loci, and a new STS, AHY1.1, was found over 250 kb from Rab1. Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species. We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16. The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B.

Plastination of stained sections of the human brain.

A method is described which combines staining of human brain tissue with subsequent plastination of the sections. Staining of 1-4 mm thick frozen sections with astra blue or aldehydefuchsin provides a sharp contrast between white and grey matter. The plastination results in sections which are durable and convenient to handle. These preparations may be used for teaching purposes in neuroanatomy and neuropathology.