BL-7010 Demonstrates Specific Binding to Gliadin and Reduces Gluten-Associated Pathology in a Chronic Mouse Model of Gliadin Sensitivity

Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.     

Enrichment of c-Met+ tumorigenic stromal cells of giant cell tumor of bone and targeting by cabozantinib

Giant cell tumor of bone (GCTB) is a very rare tumor entity, which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis, emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells, stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation, differentiation, migration, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, immunohistochemistry, antibody protein array, Alu in situ hybridization, FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies, differentiated to osteoblasts, migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4, indicating self-renewal, invasion and differentiation potential. Compared with adherent-growing cells, markers for pluripotency, stemness and cancer progression, including the CSC surface marker c-Met, were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib, an inhibitor of c-Met in phase II trials, eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met⁺ tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB.Giant cell tumor of bone (GCTB) is a very rare, osteolytic neoplasm deemed histologically benign, but it is locally aggressive and destroys bone and overlying soft tissue.^1,2 Surgery has been the preferred treatment for GCTB; however, the lesion tends to recur locally. In ~6% of cases, the development of lung metastases has been observed.^{3, 4, 5} GCTB has a predilection for the epiphyseal/metaphyseal region of long bones and the spine and thus can cause substantial morbidity.⁶ For patients with unresectable GCTB, the use of chemotherapeutics, bisphosphonates, radiation, radiofrequency thermal ablation and arterial embolization are palliative options with limited effects on tumor control.^{7, 8, 9} Recently, denosumab, a RANKL inhibitor, has been approved for GCTB, and it targets, especially the neoplastic stromal cells, which express high concentrations of RANKL.^9,10GCTB is composed of three different cell types: multinucleated, osteoclast-like giant cells, CD68⁺ phagocytic histiocytes and fibroblast-like stromal cells. The stromal cells have been identified as the neoplastic cell population,^{11, 12, 13} and it is believed that they develop from mesenchymal stem cells (MSCs).^14,15 The latter notion is supported by studies that demonstrate involvement of MSCs in tumor development—for example, in the development of sarcoma.¹⁶According to the hypothesis, cancer stem cells (CSCs) are responsible for growth, invasion, metastasis and therapy resistance of cancer, because this small sub-population within the tumor mass is thought to survive conventional cytotoxic therapy because of activated defense and survival mechanisms.¹⁷ CSCs are characterized by self-renewal potential and the ability to differentiate, thereby generating a heterogeneous cell population of the originating tumor.^{18, 19, 20} In addition, CSCs are proposed to mediate uncontrolled growth, therapy resistance, invasion and metastasis.²¹ Markers for CSCs have been identified in various tumor entities, and the selected marker-positive fractions can reconstitute the original tumor in immunodeficient mice.²² There are several surface markers for CSCs of different tumor entities and the c-Met marker represents such a typical CSC sub-population.^{23, 24, 25}c-Met belongs to the group of receptor tyrosine kinases and has a key role in cell survival, growth, angiogenesis and metastasis.²⁶ c-Met and its physiologic ligand hepatocyte growth factor (HGF) are required for normal mammalian development and have an important role in epithelial–mesenchymal interactions during organ morphogenesis.²⁶ The intracellular signaling cascades activated by c-Met include the RAS-MAPK and PI3K-AKT pathways, as well as NF-κB and Wnt/GSK-3β/β-catenin signaling.²⁶ Many carcinomas overexpress c-Met, and the surrounding stroma overexpresses HGF. Currently, the therapeutic potential of the c-Met inhibitor cabozantinib (XL184) is intensively investigated. Cabozantinib is a potent dual inhibitor of c-Met and VEGFR-2 signaling.^25,27 The clinical efficacy of cabozantinib in several progressed tumor entities is under investigation in randomized phase II studies.²⁸ At the end of 2012, cabozantinib (Cometriq) was approved by the FDA for the treatment of patients with progressive medullary thyroid carcinoma.²⁹ Cabozantinib shows promise in preventing prostate cancer spread to bone because tumors were reduced on bone scans, and bone pain decreased after patients received cabozantinib.³⁰ These data may be of importance for GCTB, but until now, cabozantinib has not been investigated for the treatment of primary bone tumors.In the present study, we demonstrate that a c-Met⁺ sub-population of low-passage stromal cells isolated from eight freshly resected GCTB specimens possess self-renewal, differentiation and migratory potential, as well as the ability to form tumors in vivo. By comparing attached-growing c-Met^low and spheroidal c-Met^high cultures, we identified enhanced pluripotency, stemness and progression, as well as the enrichment of a c-Met⁺ population. Most importantly, cabozantinib strongly inhibited the self-renewal potential and in vivo growth of GCTB stromal cells. Thus, cabozantinib may be considered an effective future therapeutic option for the targeted elimination of a tumorigenic stromal sub-population in non-resectable or recurrent GCTB.     

Agrin signalling contributes to cell activation and is overexpressed in T lymphocytes from lupus patients

It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4(+) and CD8(+) subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-alpha, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-alpha Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-alpha, and may have an important function during cell activation with potential implications for autoimmunity.     

nNOS in canine lower esophageal sphincter: colocalized with Cav-1 and Ca2+-handling proteins?

Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cav) in canine lower esophageal sphincter. The main Cav was Cav-1. It appeared to be colocalized at the cell periphery, in punctate sites, with immunoreactivity to antibodies against different COOH- and NH2-terminal epitopes of neuronal nitric oxide (NO) synthase (nNOS). One COOH-terminal-directed antibody, made in guinea pig, was used to colocalize other immunoreactivities. Those that apparently colocalized with nNOS were L-Ca2+ channels, the PM Ca2+ pump, and, in part, calreticulin and calsequestrin. The large-conductance Ca2+-activated K+ (BK(Ca)) channels were located in discrete peripheral sites, some with Cav. Immunoreactivities not fully colocalized with nNOS were to the sarcoplasmic reticulum Ca2+ pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through BK(Ca) channels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several Ca2+-handling molecules are localized at the PM with and/or near Cav. This may allow intracellular calcium concentration levels to be controlled differently than those in the cytosol near caveolae.     

Thermosensitivity of the lobster, Homarus americanus, as determined by cardiac assay

It is generally accepted that crustaceans detect, and respond to, changes in water temperature, yet few studies have directly addressed their thermosensitivity. In this investigation a cardiac assay was used as an indicator that lobsters (Homarus americanus) sensed a change in temperature. The typical cardiac response of lobsters to a 1-min application of a thermal stimulus, either warmer (n = 19) or colder (n = 17) than the holding temperature of 15 degrees C, consisted of a short bradycardia (39.5 +/- 8.0 s) followed by a prolonged tachycardia (188.2 +/- 10.7 s). Lobsters exposed to a range of rates of temperature change (0.7, 1.4, 2.6, 5.0 degrees C/min) responded in a dose-dependent manner, with fewer lobsters responding at slower rates of temperature change. The location of temperature receptors could not be determined, but lesioning of the cardioregulatory nerves eliminated the cardiac response. Although the absolute detection threshold is not known, it is conservatively estimated that lobsters can detect temperature changes of greater than 1 degree C, and probably as small as 0.15 degrees C. A comparison of winter and summer lobsters, both held at 15 degrees C for more than 4 weeks, revealed that although their responses to temperature changes were similar, winter lobsters (n = 18) had a significantly lower baseline heart rate (34.8 +/- 4.4 bpm) and a shorter duration cardiac response (174 s) than summer lobsters (n = 18; 49.9 +/- 5.0 bpm, and 320 s respectively). This suggests that some temperature-independent seasonal modulation of cardiac activity may be occurring.     

Improvement of plasmid encoded lactococcal bacteriophage resistance by mutator strain Epicurian coli XL1-Red

A random mutation strategy using mutator strain, Epicurian coli XL1-Red, was applied to a plasmid, pND018, constructed by inserting a Lactococcus lacis bacteriophage resistance gene (abiI) into a L. lactis/E. coli shuttle vector (pDL278), to introduce random mutations throughout the plasmid. Following transformation of the mutated plasmid library to a plasmid free and phage sensitive strain of L. lactis (LM0230), mutated plasmids were screened by cross-streaking and efficiency of plaquing (EOP) assays. Two strains with enhanced resistance were obtained, as well as several phage sensitive strains. Repeated transformation of the mutated plasmids to LM0230 confirmed that the observed phenotypes were caused by mutations located on the plasmids. The EOP values and plaque morphology of two enhanced phage resistance mutants were characterized at 30°C and 37°C. These results indicate that this simple procedure can be applied to generate modified plasmids with improved phage resistance, which may be of commercial value.     

Correlations of geographic distribution and temperature of embryonic development with the nuclear DNA content in the Salamandridae (Urodela, Amphibia).

We used flow cytometry to measure the nuclear DNA content in erythrocytes of 27 salamandrid species. Across these species, diploid genome size varied more than 2 fold (51.3-104.4 pg). According to genome size and geographic distribution, 3 groups of newt species were recognized: West Palearctics with smaller amounts of nuclear DNA; Nearctic, with intermediate values; and East Asiatic, with higher genome sizes. Viviparous West Palearctic salamanders differed from most of the oviparous West Palearctic newts in possessing larger genome sizes. The nuclear DNA content strongly correlates with species range limits. At the same temperature, embryos of salamandrid species with larger genome sizes have a markedly longer developmental time than those with smaller genomes. We present an analysis of the relationships between the amount of nuclear DNA and water temperature at the breeding sites.     

Use of cultivated plants and non-plant remedies for human and animal home-medication in Liubań district,Belarus

Background

To use any domestic remedy, specific knowledge and skills are required. Simple logic dictates that the use of wild plants in the context of limited interaction with nature requires prior identification, while in the case of non-plant remedies and cultivated plants this step can be omitted. This paper aims to document the current and past uses of non-plant remedies and cultivated plants in the study region for human/animal medication; to analyze the human medicinal and veterinary use areas in the context of the remedy groups; to qualitatively compare the results with relevant historical publications; and to compare the intensity and purpose of use between the remedy groups.

Methods

During field studies 134 semi-structured interviews were conducted with locals from 11 villages in the Liubań district of Belarus. Currently used home-remedies as well as those used in the past were documented by employing the folk history method. The subject was approached through health-related uses, not by way of remedies. Interview records were digitalized and structured in Detailed Use Records in order to ascertain local perceptions. An Informant Consensus Factor (FIC) was calculated for remedy groups as well as for different use categories.

Results

In the human medication area the use of nearby remedies was neither very diverse nor numerous: 266 DUR for 45 taxa belonging to 27 families were recorded for cultivated plants along with 188 DUR for 58 different non-plant remedies. The FIC values for both remedy groups were lower than for wild plants. In the ethnoveterinary medicine use area there were 48 DUR referring to the use of 14 cultivated plant taxa from 12 families and 72 DUR referring to the use of 31 non-plant remedies. The FIC value for the whole veterinary use area of cultivated plants was relatively low, yet similar to the FIC of wild plants.

Conclusions

Differences between remedy groups were pronounced, indicating that in domestic human medicine cultivated plants and non-plant remedies are either remarkably less important than wild ones or not considered worth talking about. In ethnoveterinary medicine non-plant remedies are almost equally important as wild plants, while cultivated plants are the least used. People in study area seem to still more often rely on, or are more willing to talk to strangers about, wild plants, as promoted by both official medicine and popular literature.