Loss of inhibition over master pathways of bone mass regulation results in osteosclerotic bone metastases in prostate cancer

Prostate cancer is the most common cancer among men in industrialised countries. Most patients with prostate cancer, however, will not die of it. As a result, many of them will experience symptomatic metastasis during the course of the disease. Prostate cancer has a high propensity to metastasize to bone. Unlike many other cancers prostate cancer cells induce a rather osteosclerotic than osteolytic reaction in the bone marrow by interfering with physiological bone remodelling. A proper understanding of the mechanisms of tumour cell-induced bone alterations and exaggerated bone deposition in prostate cancer may open new and urgently needed therapeutic approaches in the field of palliative care for affected patients. In this review we focus on the central role of two major regulators of bone mass, the wingless type integration site family members (WNTs) and the bone morphogenetic proteins (BMPs), in the development of osteosclerotic bone metastases.

Integrity and regeneration of mechanotransduction machinery regulate aminoglycoside entry and sensory cell death

Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23(+/+) and Cdh23(v2J/+) mice, but failed to detect GTTR uptake in Cdh23(v2J/v2J) hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23(v2J/v2J) and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.

Pathology of sarcoptic mange in red foxes (Vulpes vulpes): macroscopic and histologic characterization of three disease stages

Sarcoptic mange is a highly contagious skin disease that can have a devastating impact on affected wild mammal populations. There are notable variations in the clinical and pathologic picture of sarcoptic mange among species and among conspecifics. However, the origin of these variations is unclear. We propose a classification scheme for skin lesions associated with Sarcoptes scabiei infestation to provide a basis for a subsequent risk factor analysis. We conducted a case-control study focused on macroscopic and histologic examination of the skin, using 279 red foxes (Vulpes vulpes) found dead or shot in Switzerland between November 2004 and February 2006. All animals were submitted to gross necropsy following a detailed protocol. Selection criteria for cases (n=147) vs. controls (n=111) were the presence or absence of mange-like lesions, mite detection by isolation or histologic examination, and serologic testing for S. scabiei antibodies. Characteristic features of mange lesions were scored macroscopically in all foxes and histologically in 67 cases and 15 controls. We classified skin lesions and associated necropsy findings into three types of mange: A) early stage (n=45): focal-extensive skin lesions, thin crusts, mild to moderate alopecia, few mites, numerous eosinophils, and mild lymph node enlargement; B) hyperkeratotic, fatal form (n=86): generalized skin lesions, thick crusts with or without alopecia, foul odor, abundance of mites, numerous bacteria and yeasts, numerous lymphocytes and mast cells, severe lymph node enlargement, and emaciation; C) alopecic, healing form (n=16): focal lesions, no crusts, severe alopecia, hyperpigmentation and lichenification, absence of mites, mixed cell infiltration, and rare mild lymph node enlargement. We hypothesize that after stage A, the animal either enters stage B and dies, or stage C and survives, depending on largely unknown extrinsic or intrinsic factors affecting the host ability to control mite infestation.

Desmoplastic small round cell tumor: A rare cause of a progressive brachial plexopathy.

Introduction: Desmoplastic small round cell tumor (DSRCT) is an uncommon, embryonic-type neoplasm, typically presenting as an abdominal mass in young men. A single case of DSRCT arising in the peripheral nervous system has been reported. Methods: The clinical course, imaging, electrophysiological, intraoperative, histopathological, molecular findings, and postoperative follow-up are reported. Results: A 43-year-old man presented with slowly progressive right brachial plexopathy. Magnetic resonance imaging revealed an enlarged medial cord with heterogeneous contrast enhancement. Histology showed a "small round cell" neoplasm with a polyphenotypic immunoprofile, including epithelial and mesenchymal markers. A pathognomonic fusion of Ewing sarcoma breakpoint region 1 and Wilms tumor 1 genes (EWSR1/WT1) was present. Treatment involved gross total excision and local radiotherapy. Conclusion: Our findings confirm the occurrence of DSRCT as a primary peripheral nerve tumor. Despite its usually very aggressive clinical course, prolonged recurrence-free survival may be reached. Histomorphology and immunoprofile of DSRCT may lead to misdiagnosis as small cell carcinoma. © 2013 Wiley Periodicals, Inc.

Sudden death in a case of sickle cell anemia: post-mortem computed tomography and autopsy correlation from a radiologist's perspective

Sickle cell anemia (SCA) is a hemolytic disease characterized by the production of abnormal hemoglobin chains and distorted red blood cell morphology or sickling. "Sickle cell crisis" includes vaso-occlusive crisis, a plastic crisis, sequestration crisis, haemolytic crisis and often culminating in serious complications, organ damage and even sudden death. Post-mortem computed tomography (PMCT) findings of sickle cell disease have never been reported in literature. This case of sudden death from acute hemolytic crisis in SCA where post-mortem computed tomography (PMCT) and autopsy findings complemented each other, both revealing findings invisible to the other and both crucial to the case.

Dynamics of red blood cells in microporous membranes

We have performed microfluidic experiments with erythrocytes passing through a network of microchannels of 20–25 μm width and 5 μm of height. Red blood cells (RBCs) were flowing in countercurrent directions through microchannels connected by μm pores. Thereby, we have observed interesting flow dynamics. All pores were blocked by erythrocytes. Some erythrocytes have passed through pores, depending on the channel size and cell elasticity. Many RBCs split into two or more smaller parts. Two types of splits were observed. In one type, the lipid bilayer and spectrin network were cut at the same time. In the second type, the lipid bilayer reconnected, but the part of spectrin network stayed outside the cell forming a rope like structure, which could eventually break. The microporous membrane results in multiple breakups of the cells, which can have various clinical implications, e.g., glomerulus hematuria and anemia of patients undergoing dialysis. The cell breakup procedure is similar to the one observed in the droplet breakage of viscoelastic liquids in confinement.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Elucidation of Nucleic Acid–Drug Interactions by Tandem Mass Spectrometry

In continuation of the long tradition of mass spectrometric research at the University of Bern, our group focuses on the characterization of nucleic acids as therapeutic agents and as drug targets. This article provides a short overview of our recent work on platinated single-stranded and higher-order nucleic acids. Nearly three decades ago the development of soft ionization techniques opened a whole new chapter in the mass spectrometric analysis of not only nucleic acids themselves, but also their interactions with potential drug candidates. In contrast to modern next generation sequencing approaches, though, the goal of the tandem mass spectrometric investigation of nucleic acids is by no means the complete sequencing of genetic DNA, but rather the characterization of short therapeutic and regulatory oligonucleotides and the elucidation of nucleic acid–drug interactions. The influence of cisplatin binding on the gas-phase dissociation of nucleic acids was studied by the means of electrospray ionization tandem mass spectrometry. Experiments on native and modified DNA and RNA oligomers confirmed guanine base pairs as the preferred platination site and laid the basis for the formulation of a gas-phase fragmentation mechanism of platinated oligonucleotides. The study was extended to double stranded DNA and DNA quadruplexes. While duplexes are believed to be the main target of cisplatin in vivo, the recently discovered DNA quadruplexes constitute another promising target for anti-tumor drugs owing to their regulatory functions in the cell cycle.

How common is the lipid body-containing interstitial cell in the mammalian lung?

Pulmonary lipofibroblasts are thought to be involved in lung development, regeneration, vitamin A storage, and surfactant synthesis. Most of the evidence for these important functions relies on mouse or rat studies. Therefore, the present study was designed to investigate the presence of lipofibroblasts in a variety of early postnatal and adult mammalian species (including humans) to evaluate the ability to generalize functions of this cell type for other species. For this purpose, lung samples from 14 adult mammalian species as well as from postnatal mice, rats, and humans were investigated using light and electron microscopic stereology to obtain the volume fraction and the total volume of lipid bodies. In adult animals, lipid bodies were observed only, but not in all rodents. In all other species, no lipofibroblasts were observed. In rodents, lipid body volume scaled with body mass with an exponent b = 0.73 in the power law equation. Lipid bodies were not observed in postnatal human lungs but showed a characteristic postnatal increase in mice and rats and persisted at a lower level in the adult animals. Among 14 mammalian species, lipofibroblasts were only observed in rodents. The great increase in lipid body volume during early postnatal development of the mouse lung confirms the special role of lipofibroblasts during rodent lung development. It is evident that the cellular functions of pulmonary lipofibroblasts cannot be transferred easily from rodents to other species, in particular humans.

Magnetic resonance imaging features of an extranodal T-cell rich B-cell lymphoma in the pharyngeal mucosa in a horse

An 11-year-old Warmblood gelding was presented for inspiratory stridor and dysphagia. Based on history and clinical examination, a solitary mass localised in the oropharynx was suspected. Due to its inaccessibility and defensive behaviour of the horse, it was difficult to visualise this mass either by upper airway endoscopy or by oral examination and the conventional imaging methods (radiology and ultrasound) provided only limited information. Fine needle aspiration cytology was suggestive of lymphoma, but the exact localisation and the extent of tissue infiltration of the tumour could only be defined by magnetic resonance imaging (MRI). MRI has proved to be a very useful diagnostic tool in equine lameness investigation and, as this case illustrates, it has considerable diagnostic potential for soft tissue examination of the equine head.

Cell cycle-dependent phosphorylation of Theileria annulata schizont surface proteins.

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.

Elevated levels of endocannabinoids in chronic hepatitis C may modulate cellular immune response and hepatic stellate cell activation.

The endocannabinoid (EC) system is implicated in many chronic liver diseases, including hepatitis C viral (HCV) infection. Cannabis consumption is associated with fibrosis progression in patients with chronic hepatitis C (CHC), however, the role of ECs in the development of CHC has never been explored. To study this question, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were quantified in samples of HCV patients and healthy controls by gas and liquid chromatography mass spectrometry. Fatty acid amide hydrolase (FAAH) and monoaclyglycerol lipase (MAGL) activity was assessed by [3H]AEA and [3H]2-AG hydrolysis, respectively. Gene expression and cytokine release were assayed by TaqMan PCR and ELISpot, respectively. AEA and 2-AG levels were increased in plasma of HCV patients, but not in liver tissues. Hepatic FAAH and MAGL activity was not changed. In peripheral blood mononuclear cells (PBMC), ECs inhibited IFN-γ, TNF-α, and IL-2 secretion. Inhibition of IL-2 by endogenous AEA was stronger in PBMC from HCV patients. In hepatocytes, 2-AG induced the expression of IL-6, -17A, -32 and COX-2, and enhanced activation of hepatic stellate cells (HSC) co-cultivated with PBMC from subjects with CHC. In conclusion, ECs are increased in plasma of patients with CHC and might reveal immunosuppressive and profibrogenic effects.

Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen. Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet. ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry.

Effect of bone graft density on in vitro cell behavior with enamel matrix derivative

OBJECTIVES Bone replacement grafting materials play an important role in regenerative dentistry. Despite a large array of tested bone-grafting materials, little information is available comparing the effects of bone graft density on in vitro cell behavior. Therefore, the aim of the present study is to compare the effects of cells seeded on bone grafts at low and high density in vitro for osteoblast adhesion, proliferation, and differentiation. MATERIALS AND METHODS The response of osteoblasts to the presence of a growth factor (enamel matrix derivative, (EMD)) in combination with low (8 mg per well) or high (100 mg per well) bone grafts (BG; natural bone mineral, Bio-Oss®) density, was studied and compared for osteoblast cell adhesion, proliferation, and differentiation as assessed by real-time PCR. Standard tissue culture plastic was used as a control with and without EMD. RESULTS The present study demonstrates that in vitro testing of bone-grafting materials is largely influenced by bone graft seeding density. Osteoblast adhesion was up to 50 % lower when cells were seeded on high-density BG when compared to low-density BG and control tissue culture plastic. Furthermore, proliferation was affected in a similar manner whereby cell proliferation on high-density BG (100 mg/well) was significantly increased when compared to that on low-density BG (8 mg/well). In contrast, cell differentiation was significantly increased on high-density BG as assessed by real-time PCR for markers collagen 1 (Col 1), alkaline phosphatase (ALP), and osteocalcin (OC) as well as alizarin red staining. The effects of EMD on osteoblast adhesion, proliferation, and differentiation further demonstrated that the bone graft seeding density largely controls in vitro results. EMD significantly increased cell attachment only on high-density BG, whereas EMD was able to further stimulate cell proliferation and differentiation of osteoblasts on control culture plastic and low-density BG when compared to high-density BG. CONCLUSION The results from the present study demonstrate that the in vitro conditions largely influence cell behavior of osteoblasts seeded on bone grafts and in vitro testing. CLINICAL RELEVANCE These results also illustrate the necessity for careful selection of bone graft seeding density to optimize in vitro testing and provide the clinician with a more accurate description of the osteopromotive potential of bone grafts.