Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2.

The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.

[Rare erosive arthritis and dermatitis syndrome in Whipple's disease]. / Seltenes erosives Arthritis- und Dermatitissyndrom bei Morbus Whipple.

Whipple's disease is a rare infectious disease, which can affect various organ systems. Arthritis is a common symptom and therefore the infection is often misdiagnosed as seronegative rheumatoid arthritis. In rare cases an infection with Tropheryma whipplei can also cause skin lesions, such as subcutaneous nodules, erythema nodosum or vasculitis. This article reports the case of a 77-year-old female patient with erosive joint changes, persistently elevated serological inflammation markers and recurrent ulcerative lesions of the lower extremities, which were initially misdiagnosed as rheumatoid vasculitis. In cases of a clinically suspected infection with Tropheryma whipplei an early biopsy of the affected organ system is essential for the diagnosis.

Improvement of ELISA procedures through simultaneous addition of antigen and detection antibody and elimination of washing steps.

The enzyme-linked immunosorbent assay (ELISA) is extensively used for measurement of proteins utilized for various research and diagnostic purposes in the biological disciplines. However, it is a labor-intensive and lengthy procedure due to a number of incubation and washing steps required for performing the assay. The ELISA procedure in the current study has been simplified through the simultaneous addition of antigen and detection antibody and elimination of washing steps. This resulted in a decreased time required to perform the procedure and without affecting assay capability. This approach offers the possibility of increasing ELISA productivity in low throughput laboratories without the need for alternative analytical platforms which would require significant assay redevelopment and capital expense.

[Gastrointestinal stromal tumours (GIST)--development in pathology, surgery and medical therapy. Developed during the 10th German GIST-meeting, Göttingen]. / Gastrointestinale Stromatumoren (GIST)--Neues zu Pathologie, Chirurgie und medikamentöser Therapie. Erarbeitet im Rahmen des 10. Deutschen GIST-Treffens, Göttingen.

The first description of ligand-independent activating mutations in the KIT gene, which encodes the tyrosine-kinase KIT, greatly improved our understanding of gastrointestinal stromal tumour (GIST) biology. The therapeutic success in GIST has made tyrosine kinase inhibitors a "paradigm of targeted therapy". Deciphering resistance mechanisms in GIST has had implications for many other kinase-driven cancers. To exchange current knowledge within the field of GIST, the German GIST Meeting has taken place for now 10 years, traditionally in Göttingen. Subjects discussed include clinical diagnostics, pathology, surgery, and medical therapy. The following presentation gives an overview of the last meeting held in December 2013, including distinctive features in GIST and current data on the different topics.

Evaluation of single-cell biomechanics as potential marker for oral squamous cell carcinomas: a pilot study.

OBJECTIVES: Early detection of oral cancer is a major health issue. The objective of this pilot study was to analyze the deformability of healthy and cancer cells using a microfluidic optical stretcher (OS). MATERIAL AND METHODS: Different cancer cell lines, primary oral cancer cells, and their healthy counterparts were cultivated and characterized, respectively. A measurable deformation of the cells along the optical axis was detected, caused by surface stress, which is optically induced by the laser power. RESULTS: All cells revealed a viscoelastic extension behavior and showed a characteristic deformation response under laser light exposure. The CAL-27/-33 cells exhibited the highest relative deformation. All other cells achieved similar values, but on a lower level. The cytoskeleton reacts sensitively of changing environmental conditions, which may be influenced by growth behavior of the cancer specimens. Nevertheless, the statistical analysis showed significant differences between healthy and cancer cells. CONCLUSION: Generally, malignant and benign cells showed significantly different mechanical behavior. Cancer-related changes influence the composition of the cytoskeleton and thus affect the deformability, but this effect may be superimposed by cell cultivation conditions or cell doubling time. These influences had to be substituted by brush biopsies to minimize confounders in pursuing investigations.

Emx2 is a dose-dependent negative regulator of Sox2 telencephalic enhancers.

The transcription factor Sox2 is essential for neural stem cells (NSC) maintenance in the hippocampus and in vitro. The transcription factor Emx2 is also critical for hippocampal development and NSC self-renewal. Searching for 'modifier' genes affecting the Sox2 deficiency phenotype in mouse, we observed that loss of one Emx2 allele substantially increased the telencephalic ß-geo (LacZ) expression of a transgene driven by the 5' or 3' Sox2 enhancer. Reciprocally, Emx2 overexpression in NSC cultures inhibited the activity of the same transgene. In vivo, loss of one Emx2 allele increased Sox2 levels in the medial telencephalic wall, including the hippocampal primordium. In hypomorphic Sox2 mutants, retaining a single 'weak' Sox2 allele, Emx2 deficiency substantially rescued hippocampal radial glia stem cells and neurogenesis, indicating that Emx2 functionally interacts with Sox2 at the stem cell level. Electrophoresis mobility shift assays and transfection indicated that Emx2 represses the activities of both Sox2 enhancers. Emx2 bound to overlapping Emx2/POU-binding sites, preventing binding of the POU transcriptional activator Brn2. Additionally, Emx2 directly interacted with Brn2 without binding to DNA. These data imply that Emx2 may perform part of its functions by negatively modulating Sox2 in specific brain areas, thus controlling important aspects of NSC function in development.

Liver resection for hepatocellular carcinoma in patients without cirrhosis.

BACKGROUND: Data on liver resection for hepatocellular carcinoma (HCC) without cirrhosis are sparse. The present study was conducted to evaluate the indications and results of liver resection for HCC with regard to safety and efficacy. METHODS: Data for patients who had liver resection for HCC without cirrhosis between January 1996 and March 2011 were retrieved retrospectively using a prospective database containing information on all patients who underwent hepatectomy for HCC. Patient and tumour characteristics were analysed for influence on overall and disease-free survival to identify prognostic factors by univariable and multivariable analysis. RESULTS: The 1-, 3- and 5-year overall survival rates after resection with curative intent for HCC without cirrhosis were 84, 66 and 50 per cent respectively. Disease-free survival rates were 69, 53 and 42 per cent respectively. The 90-day mortality rate was 4·5 per cent (5 of 110 patients). Surgical radicality and growth pattern of the tumour were independent prognostic factors for overall survival. Disease-free survival after resection with curative intent was independently affected by growth pattern and by the number and size of tumour nodules. CONCLUSION: Liver resection for HCC without cirrhosis carries a low perioperative risk and excellent long-term outcome if radical resection is achieved.

[Large adenomas of the colon and rectum--specific features of the diagnostics]. / Das große Adenom des Kolons und Rektums--Besonderheiten der Diagnostik.

Carcinomas mostly originate from preneoplastic lesion of different origin, namely serrated and non-serrated adenomas. The probability of malignant transformation correlates to the size of the adenoma. The determining diagnostic method is colonoscopy with collection of tissue samples or endoscopic biopsies for histological investigations. For the necessary identification of the pathology some specific features of the treatment are to be followed. In future, other information, such as for example, molecular characteristics are expected from carcinoma pathways. Premalignant and malignant changes carry a row of DNA changes (e.g., a mutation in K-ras proto-oncogen). The 7th edition of the TNM classification of colorectal tumours shows minor changes in the T-, N- and M-categories and in stage grouping. It remains to be seen how far the classification into sm1 ti sm3 will be taken into consideration.

An improved Fc function assay utilizing CMV antigen-coated red blood cells generated with synthetic function-spacer-lipid constructs.

BACKGROUND AND OBJECTIVES: The Fc function assessment of immunoglobulin products is performed to confirm that the biological activity of immunoglobulin products is not affected by manufacturing and storage conditions. The European Pharmacopoeia (EP) test is cumbersome and time-consuming especially with respect to the derivatization of red blood cells (RBC) with rubella antigen via tanninization. We investigated the KODE biosurface engineering technology for inserting specific antigens into red blood cell membranes to create kodecytes and evaluated their suitability for use in the Fc function assay. MATERIALS AND METHODS: Human RBC were derivatized with function-spacer-lipid (FSL) constructs comprising of a peptide epitope of a cytomegalovirus (CMV) surface protein, conjugated to a spacer and lipid tail. These kodecytes were used in an Fc function assay based on the monitoring of complement-mediated haemolysis of immunoglobulin-coated red cells. Optimization of FSL construct, immunoglobulin and complement concentration was undertaken. Fc values obtained for various immunoglobulin batches were compared with those from the EP-based method. Carbohydrate-bearing FSLs Galili (Galα1-3Galß1-4GlcNAcß), Galα1-4GlcNAcß and GalNAcα1-3Galß were also evaluated. RESULTS: Fc function indices determined with CMV peptide construct-coated red cells were comparable to those obtained with the EP-based method with respect to specificity and precision. Carbohydrate-bearing FSLs revealed Fc indices lower than expected. CONCLUSION: The use of CMV kodecytes was shown to be a convenient means of generating red cells for the determination of Fc function of immunoglobulin products and offers the possibility of significantly reducing the time required to perform this assay.

[Laryngeal MALT lymphoma with known Sjögren syndrome]. / MALT-Lymphom des Larynx bei bekanntem Sjögren-Syndrom.

Hematopoietic neoplasms represent about 1% of all laryngeal neoplasms. MALT lymphoma arises from mucosa-associated lymphoid tissue and is associated with chronic inflammatory disease. Patients with Sjögren syndrome have a higher risk for development of non-Hodgkin lymphoma (MALT lymphoma). To date, only cases of MALT lymphoma of the salivary glands, thymus and stomach associated with Sjögren syndrome have been published. We present the case of a MALT lymphoma of the larynx associated with Sjögren syndrome. Radiation and chemotherapy are the first line of therapy as published in the literature.

An improved method for the determination of Fc function of immunoglobulins.

BACKGROUND AND OBJECTIVES: The Fc function assay of immunoglobulin G is performed to ensure that the biological activity of the molecule is not compromised during purification and storage. The current British Pharmacopoeia (BP) method is cumbersome, time consuming and requires a continuous source of fresh red blood cells. We have modified the technique to improve the convenience, throughput and reproducibility of the assay by using frozen red blood cells, a microtitre plate format and electronic derivative analysis for processing results. MATERIALS AND METHOD: Immunoglobulin G samples were assayed using either the BP Fc function procedure or a new procedure incorporating frozen cells, microtitre format and electronic derivative analysis. RESULTS: The Fc function results demonstrated that there was no significant difference between the BP and the new improved procedure. CONCLUSION: In this study, we demonstrated that a new Fc function assay incorporating frozen red blood cells, microtitre format and derivative analysis of haemolysis curves has been shown to be comparable to the standard BP procedure. It offers a more convenient and quicker means of performing analysis of Fc function of immunoglobulins.

Studies on basic fibroblast growth factor (FGF-beta) gene expression in the rat and pig ovary using in situ hybridization and quantitative reverse transcriptase--polymerase chain reaction techniques.

In order to gain further understanding of the physiology of basic fibroblast growth factor (FGF-beta) in the mammalian reproductive tract, the expression of FGF-beta mRNA in the rat and porcine ovary has been examined by in situ hybridization and quantitative reverse transcriptase-polymerase chain reaction techniques during different stages of the estrus cycle. The results confirm that an increase in FGF-beta mRNA levels occurs over the course of the estrus cycle. No FGF-beta gene expression was detected during diestrus, the non-hormonal phase of the cycle, or at the early proestrus stage of the cycle. During late proestrus and estrus, FGF-beta mRNA was predominantly localized to granulosa cells of the dominant follicles, and to a lesser extent, to secondary antral follicles not committed to ovulation. These cells also expressed FGF-beta mRNA during this phase of follicular development, albeit in low abundance. During metestrus, after ovulation, in the newly formed corpora lutea FGF-beta mRNA levels were maximal, however on entering the next cycle commencing at diestrus, no FGF-beta mRNA was observed in the degenerating corpora lutea. These results indicate that expression of the FGF-beta gene is differentially regulated during the estrus cycle. The biological significance of this expression and the potential role of FGF-beta in local intra-ovarian regulation of the repetitive cycles of follicular differentiation, proliferation and maturation associated with ovarian revascularization are discussed.

Isolation, characterisation and tissue localisation of an N-terminal-truncated variant of fibroblast growth factor.

An additional form of basic fibroblast growth factor (FGF), possessing full biological activity, has been isolated from bovine pituitary extracts. This polypeptide mitogen exhibits an apparent molecular weight of 17,500 by SDS-PAGE and lacks the first four N-terminal amino acid residues (Pro-Ala-Leu-Pro) of a previously described FGF preparation. Polyclonal antibodies have been raised to this FGF variant and used to examine the distribution of immunoreactive FGF species in rat and bovine tissues by immunoblot procedures. In extracts of rat pituitary, kidney, spleen and liver tissues several higher molecular weight species were detected with immunoreactive 70 kDa and 27 kDa protein bands predominating whilst a 32 kDa immunoreactive protein was also present in the brain. In bovine tissues several immunoreactive proteins with molecular weights greater than 17,500 were also observed in the brain, pituitary, adrenal, ovary and serum with these immunoblot procedures. With bovine pituitary extracts, six immunoblot bands corresponding to immunoreactive proteins of molecular weights 80,000-14,200 were observed, whilst with the brain extracts four immunoblot bands and with serum eight immunoblot bands ranging from 150,000 to 14,200 were detected. With non-immune rabbit serum, no polypeptides/proteins of corresponding molecular weight could be detected with similar immunoblot procedures. The possible relationships of these immunoreactive proteins to FGF precursor forms at various stages of processing are described.

Effect of intraperitoneally, intravenously and intralesionally administered monoclonal anti-beta-FGF antibodies on rat chondrosarcoma tumor vascularization and growth.

The growth and vascularization of many tumours has been reported to be associated with the overexpression of the potent mitogenic and angiogenic polypeptide basic fibroblast growth factor (beta-FGF). Consequently, it has been proposed that inhibition of beta-FGF action would prevent the growth of beta-FGF-dependent tumours. In this study, cell culture assays were established to assess the ability of mouse monoclonal DG-2 anti-beta-FGF antibodies to inhibit the mitogenic action of beta-FGF in vitro. Following in vitro characterisation, the monoclonal DG-2 antibodies were used to evaluate the role of beta-FGF in promoting the vascularization and growth of rat chondrosarcoma tumours. The effect the monoclonal anti-B-FGF antibodies had on tumour vascularization and growth in vivo were monitored using a 99m Technetium (99mTc)-labelled red blood cell procedure. The characterization studies confirmed that the DG-2 monoclonal antibody recognised beta-FGF and inhibited its mitogenic action on mouse Balb/c cells and bovine endothelial cells in vitro. When examined in vivo, intralesional administration of mouse monoclonal DG-2 antibody significantly inhibited rat chondrosarcoma growth and vascularization. However when the monoclonal DG-2 antibody was administered intraperitoneally or intravenously no attenuation of rat chondrosarcoma tumour vascularization or growth was observed. This report has confirmed the potential effectiveness of anti-beta-FGF antibodies in the regulation of tumour growth. It has also demonstrated that further studies on the pharmacokinetics of administered antibodies and their mode of delivery are required so that the effectiveness of such anti-growth factor immunotherapy can be assured.

Detection of FGF-beta mRNA in chondrosarcoma cells by a new in situ hybridization technique with synthetic oligonucleotide probes.

Fibroblast growth factor beta (FGF-beta) is a potent mitogenic and angiogenic factor produced by a large number of normal and transformed cells. In this paper we report a new application of the in situ hybridization procedure which has allowed the detection of FGF-beta mRNA in chondrosarcoma cells using 35S-labelled synthetic oligonucleotide probes.

A new quantitative polymerase chain reaction-high performance ion exchange liquid chromatographic method for the detection of fibroblast growth factor-beta (FGF-beta) gene amplification.

A method for the quantitative determination of fibroblast growth factor-beta (FGF-beta) genomic amplification based on the use of optimized polymerase chain reaction (PCR) procedures and high performance ion exchange (HPIEX) liquid chromatography has been developed. Co-amplification of a second genomic species permits internal standardization of the techniques during optimization of the reaction conditions, the PCR cycle number and the PCR cycle efficiency, as well as during the analytical HPIEX chromatographic determination of the PCR products. These investigations confirm the versatility of these procedures to quantitatively analyse FGF-beta gene amplification in various cells and tissues.

The effect of intervertebral disc space narrowing on the contact force between the nerve root and a simulated disc protrusion.

An instrumented probe mounted on the anterior surface of the lumbar spine over an excised lumbar intervertebral disc was used to stimulate a disc protrusion in 12 fresh cadavers. The contact force between probe and nerve root was measured as a function of two independent variables: probe protrusion depth and disc space height. The contact force on the nerve root was found to increase with increasing probe depth. Disc space widening increased the contact force while narrowing the disc space decreased it. A simple mechanical model analysis confirmed that the force exerted on the nerve root by the probe is the result of tension produced in the nerve root as it is deformed by the probe. The mechanical principle that disc narrowing can reduce the pressure on a nerve root produced by a disc protrusion may be an explanation of how chemonycleolysis relieves sciatic pain.

The red eye.

The red eye is a clinical problem encountered on a daily basis in most emergency departments. A careful history and focused ophthalmologic examination will lead to a correct diagnosis and proper therapy. The red eye without photophobia, pain, or visual disturbance is most commonly a result of conjunctivitis. The presence of any of these symptoms indicates a need to investigate for a more serious cause. Infectious causes of conjunctivitis should always be investigated, but allergies and hypersensitivity also should be considered if the history is appropriate. Emergency department treatment of the red eye should only include corticosteroids when the diagnosis is certain and ophthalmologic consultation is obtained.