A multicenter double-blind, placebo-controlled randomized trial to evaluate the safety and efficacy of bovine colostrum in the treatment of severe alcoholic hepatitis (SAH).

BACKGROUND: Severe alcoholic hepatitis (SAH) is associated with high mortality. Numerous studies and meta-analysis have reported that corticosteroids reduce the 28-day mortality in SAH, but not the 6-month mortality. Therefore, newer treatments for SAH need to be studied. A pilot study from our group had recently treated ten patients with SAH with bovine colostrum (BC) [20 g thrice in a day for 8 weeks] and prednisolone. This therapy improved the biological functions and 3-month mortality. However, as more and more data showed the failure of corticosteroids to improve the 3- and 6-month mortality, especially in patients with high mDF and MELD scores, we planned this trial to study the safety and efficacy of BC (without corticosteroids) in the treatment of SAH. METHOD: This is a multicenter, parallel, double-blind, randomized (1:1) placebo-controlled trial, which will enroll 174 patients with SAH from 5 academic centers in the India. Patients will receive freeze-dried BC or placebo by random 1:1 allocation for 4 weeks. The primary outcome measure is survival at 3 months. The secondary outcome measures are survival at 1 month, change in mDF and MELD scores, change in endotoxin and cytokines (alpha TNF, IL6, and IL8) levels, number of episodes of sepsis [pneumonia, spontaneous bacterial peritonitis (SBP), cellulitis, urinary tract infection (UTI)] from baseline to 4 weeks. DISCUSSION: This study will evaluate the safety and efficacy of bovine colostrum in improving the survival of patients with SAH. TRIAL REGISTRATION: ClinicalTrials.gov NCT02473341. Prospectively registered on June 16, 2015.

High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression.

Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and develop a general strategy for enhancing the affinity of chromodomains of the human Cbx protein family. Our strategy allows us to develop powerful probes for genome-wide binding analysis and live-cell imaging. Furthermore, we use optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing.

A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family.

The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease. Using phage-displayed synthetic antibody libraries, we have conducted selections against 12 human IFNα subtypes to explore our ability to obtain fine-specificity antibodies that recognize and antagonize the biological signals induced by a single IFNα subtype. For the first time, we have isolated antibodies that specifically recognize individual IFNα subtypes (IFNα2a/b, IFNα6, IFNα8b and IFNα16) with high affinity that antagonize signaling. Our results show that highly specific antibodies capable of distinguishing between closely related cytokines can be isolated from synthetic libraries and can be used to characterize cytokine abundance and function.

Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma.

Intratumoral heterogeneity (ITH) has increasingly being described for multiple cancers as the root cause of therapy resistance. Recent studies have started to explore the scope of ITH in glioblastoma (GBM), a highly aggressive and fatal form of brain tumor, to explain its inevitable therapy resistance and disease relapse. In this review, we detail the emerging data that explores the extensive genetic, cellular and functional ITH present in GBM. We discuss current experimental models of human GBM recurrence and suggest harnessing new technologies (CRISPR-Cas9 screening, CyTOF, cellular barcoding, single cell analysis) to delineate GBM ITH and identify treatment-refractory cell populations, thus opening new therapeutic windows. We will also explore why current therapeutics have failed in clinical trials and how ITH can inform us on developing empiric therapies for the treatment of recurrent GBM.

Sorafenib induced hepatic encephalopathy.

A 60 year old male, known case of Hepatitis C related cirrhosis was diagnosed with exophytic Hepatocellular carcinoma (size 2.1 x 2.2 cm), Barcelona Clinic Liver Cancer Stage A, on routine surveillance. He refused liver Transplant and underwent laparoscopic segmental resection. Thereafter patient was started on Tablet Sorafenib 400mg twice daily to prevent recurrence of Hepatocellular carcinoma. On 18st post-operative day, patient presented with Hepatic encephalopathy. Routine investigations and MRI Brain were normal; Venous ammonia was high. Sorafenib was discontinued, and neurological symptoms resolved within 24 hours. The ammonia level decreased from 112 to 30 µmol/L. Hepatic encephalopathy recurred 14 days after Sorafenib reintroduction at a dose of 400 mg / day. It resolved within 24 hours of withdrawal of Sorafenib. Sorafenib induced recurrent acute overt Hepatic encephalopathy with biochemical corroboration is reported here.

Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack.

The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported.

Can taxanes provide benefit in patients with CNS tumors and in pediatric patients with tumors? An update on the preclinical development of cabazitaxel.

PURPOSE: While first-generation taxanes are valuable treatment options for many solid tumors, they are limited by an inability to cross the blood-brain barrier (BBB) and by limited efficacy in pediatric patients. Following promising preclinical data for the next-generation taxane cabazitaxel, including activity in tumor models fully sensitive, poorly sensitive or insensitive to docetaxel, and its ability to cross the BBB, further preclinical studies of cabazitaxel relevant to these two clinical indications were performed. METHODS: Cabazitaxel brain distribution was assessed in mice, rats and dogs. Cabazitaxel antitumor activity was assessed in mice bearing intracranial human glioblastoma (SF295; U251) xenografts, and subcutaneous cell line-derived human pediatric sarcoma (rhabdomyosarcoma RH-30; Ewing's sarcoma TC-71 and SK-ES-1) or patient-derived pediatric sarcoma (osteosarcoma DM77 and DM113; Ewing's sarcoma DM101) xenografts. The activity of cabazitaxel-cisplatin combination was evaluated in BALB/C mice bearing the syngeneic murine colon adenocarcinoma, C51. RESULTS: Cabazitaxel penetrated rapidly in the brain, with a similar brain-blood radioactivity exposure relationship across different animal species. In intracranial human glioblastoma models, cabazitaxel demonstrated superior activity to docetaxel both at early (before BBB disruption) and at advanced stages, consistent with enhanced brain penetration. Compared with similar dose levels of docetaxel, cabazitaxel induced significantly greater tumor growth inhibition across six pediatric tumor models and more tumor regressions in five of the six models. Therapeutic synergism was observed between cisplatin and cabazitaxel, regardless of administration sequence. CONCLUSIONS: These preclinical data suggest that cabazitaxel could be an effective therapy in CNS and pediatric tumors, supporting ongoing clinical evaluation in these indications.

Secreted semaphorin 5A suppressed pancreatic tumour burden but increased metastasis and endothelial cell proliferation.

BACKGROUND: Our earlier reports demonstrated that membrane-bound semaphorin 5A (SEMA5A) is expressed in aggressive pancreatic cancer cells and tumours, and promotes tumour growth and metastasis. In this study, we examine whether (1) pancreatic cancer cells secrete SEMA5A and (2) that secreted SEMA5A modulates certain phenotypes associated with tumour progression, angiogenesis and metastasis through various other molecular factors and signalling proteins. METHODS AND RESULTS: In this study, we show that human pancreatic cancer cell lines secrete the extracellular domain (ECD) of SEMA5A (SEMA5A-ECD) and overexpression of mouse Sema5A-ECD in Panc1 cells (not expressing SEMA5A; Panc1-Sema5A-ECD; control cells - Panc1-control) significantly increases their invasion in vitro via enhanced ERK phosphorylation. Interestingly, orthotopic injection of Panc1-Sema5A-ECD cells into athymic nude mice results in a lower primary tumour burden, but enhances the micrometastases to the liver as compared with Panc1-control cells. Furthermore, there is a significant increase in proliferation of endothelial cells treated with conditioned media (CM) from Panc1-Sema5A-ECD cells and a significant increase in microvessel density in Panc1-Sema5A-ECD orthotopic tumours compared with those from Panc1-control cells, suggesting that the increase in liver micrometastases is probably due to increased tumour angiogenesis. In addition, our data demonstrate that this increase in endothelial cell proliferation by Sema5A-ECD is mediated through the angiogenic molecules - interleukin-8 and vascular endothelial growth factor. CONCLUSION: Taken together, these results suggest that a bioactive, secreted form of Sema5A-ECD has an intriguing and potentially important role in its ability to enhance pancreatic tumour invasiveness, angiogenesis and micrometastases.

Synthetic antibodies: concepts, potential and practical considerations.

The last 100 years of enquiry into the fundamental basis of humoral immunity has resulted in the identification of antibodies as key molecular sentinels responsible for the in vivo surveillance, neutralization and clearance of foreign substances. Intense efforts aimed at understanding and exploiting their exquisite molecular specificity have positioned antibodies as a cornerstone supporting basic research, diagnostics and therapeutic applications [1]. More recently, efforts have aimed to circumvent the limitations of developing antibodies in animals by developing wholly in vitro techniques for designing antibodies of tailored specificity. This has been realized with the advent of synthetic antibody libraries that possess diversity outside the scope of natural immune repertoires and are thus capable of yielding specificities not otherwise attainable. This review examines the convergence of technologies that have contributed to the development of combinatorial phage-displayed antibody libraries. It further explores the practical concepts that underlie phage display, antibody diversity and the methods used in the generation of and selection from phage-displayed synthetic antibody libraries, highlighting specific applications in which design approaches gave rise to specificities that could not easily be obtained with libraries based upon natural immune repertories.

A protective role for periostin and TGF-ß in IgE-mediated allergy and airway hyperresponsiveness.

BACKGROUND: The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up-regulated in the airway epithelium of asthmatics and to increase active TGF-ß. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown. OBJECTIVE: To determine the effect of periostin deficiency on airway responses to inhaled allergen. METHODS: In vivo measures of airway responsiveness, inflammation, and remodelling were made in periostin deficient mice and wild-type controls following repeated intranasal challenge with Aspergillus fumigatus antigen. In vitro studies of the effects of epithelial cell-derived periostin on murine T cells were also performed. RESULTS: Surprisingly, compared with wild-type controls, periostin deficient mice developed increased AHR and serum IgE levels following allergen challenge without differences in two outcomes of airway remodelling (mucus metaplasia and peribronchial fibrosis). These changes were associated with decreased expression of TGF-ß1 and Foxp3 in the lungs of periostin deficient mice. Airway epithelial cell-derived periostin-induced conversion of CD4(+) CD25(-) cells into CD25(+) , Foxp3(+) T cells in vitro in a TGF-ß dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Allergen-induced increases in serum IgE and bronchial hyperresponsiveness are exaggerated in periostin deficient mice challenged with inhaled aeroallergen. The mechanism of periostin's effect as a brake on allergen-induced responses may involve augmentation of TGF-ß-induced T regulatory cell differentiation.

EMMPRIN regulates the canonical Wnt/beta-catenin signaling pathway, a potential role in accelerating lung tumorigenesis.

Advances in the field of tumor biology have identified that tumor cells co-opt developmental signaling pathways of embryonic stem cells and thus gain the ability to proliferate, differentiate and alter cell-cell interactions. One such pathway is the Wnt/beta-catenin signaling pathway. High levels of EMMPRIN expression have been shown to correlate with poor prognosis and metastasis in a broad range of tumors. Although a variety of functions are attributed to EMMPRIN in tumorigenesis, the specific mechanism(s) through which it can exert its effects have not been elucidated, until now. In this study, we identify EMMPRIN as a novel regulator of the canonical Wnt/beta-catenin signaling pathway in lung cancer. Increasing EMMPRIN expression levels in lung cancer epithelial cells upregulated the beta-catenin signaling pathway and silencing EMMPRIN inhibited beta-catenin signaling, cell migration, proliferation, anchorage-independent growth and tumor growth in a mouse tumor xenograft model. These results provide a compelling rationale for targeting EMMPRIN for anticancer therapies. Understanding the molecular mechanisms driving EMMPRIN-induced lung tumorigenesis will provide enormous benefits in developing new therapeutic treatments for this and other forms of cancer.

Monitoring of Cd pollution in soils and plants irrigated with untreated sewage water in some industrialized cities of Punjab, India.

The disposal of industrial and sewage water is a problem of increasing importance throughout the world. In India, and most of the developing countries untreated sewage and industrial wastes are discharged on land or into the running water streams which is used for irrigating crops. These wastes often contain high amount of trace elements which may accumulate in soils in excessive quantities on long term use and enter the food chain through absorption by the plants. Among the trace metals, Cd has received the greater attention because of its easy absorption and accumulation in plants and animals to levels toxic for their health. The objective of this study conducted in three industrially different cities viz., Ludhiana, Jalandhar and Malerkotla was to monitor the extent of Cd accumulation in soils and plants receiving untreated sewage water. Plant and soil samples were collected from sewage and tubewell irrigated areas. Soil samples were analysed for texture, pH, EC, organic carbon (OC), CaCO(3), bioavailable DTPA-Cd and plant samples were analysed for total Cd. In sewage irrigated soils, the mean values of pH were lower but organic carbon and electrical conductivity were generally higher both in surface and sub-surface layers of all the three cities as compared to tubewell irrigated soils. The mean DTPA- extractable Cd in sewage irrigated soil was 6.3- and 4.36-fold in Ludhiana, 3.38- and 1.71-fold in Jalandhar and 3.35- and 6.67-fold in Malerkotla in 0-15 and 15-30 cm soil depth, respectively, compared with the values in tubewell irrigated soils. The accumulation of DTPA-Cd in sewage irrigated soils was restricted to 30 cm depth after which the values were generally close to values in tubewell irrigated soils. Soil pH, OC, CaCO(3), clay and silt collectively accounted for 37.1%, 65.1% and 53.9% DTPA-extractable bioavailable Cd in soils of Ludhiana, Jalandhar and Malerkotla, respectively. Lower R(2) values in Ludhiana suggest that factors other than the ones mentioned may be affecting Cd availability. At all sites plants receiving sewage irrigation had elevated levels of Cd as compared to the plants receiving tubewell irrigation. The mean Cd content of sewage irrigated plants irrespective of the city was 5.96 microg g(-1) dry matter as compared to 0.98 microg g(-1) dry matter in tubewell irrigated plants. The results suggest that the intake of Cd obtained from consumption of crops grown on sewage irrigated soils would be much higher than the tolerable limits set by WHO and may, therefore, prove potentially toxic leading to various health ailments to humans and animals.

Exposure of human corneal epithelial cells to contact lenses in vitro suppresses the upregulation of human beta-defensin-2 in response to antigens of Pseudomonas aeruginosa.

Bacterial keratitis is a sight-threatening complication of contact lens wear, and Pseudomonas aeruginosa is a commonly isolated pathogen. The mechanisms by which lenses predispose the cornea to P. aeruginosa infection are unknown. Corneal epithelial cells express numerous innate defenses, some of which have bactericidal effects against P. aeruginosa. One of these is human beta-defensin-2 (hBD-2), which is upregulated in response to lipopolysaccharide or flagellin antigens. We hypothesized that prior exposure of corneal epithelia to a contact lens would interfere with upregulation of hBD-2 in response to P. aeruginosa. A novel in vitro model was used in which cultured human corneal epithelial cells were exposed to a hydrophilic contact lens for up to 3.5 days prior to challenge with a culture supernatant of P. aeruginosa antigens for 6h. Without prior lens exposure, the supernatant caused >2-fold upregulation of hBD-2 mRNA message and expression of hBD-2 peptide. Prior contact lens exposure blocked this upregulation without obvious effects on cell health. Western immunoblot and luciferase reporter studies showed that Pseudomonas-induced hBD-2 upregulation involved MyD88, c-Jun N-terminal kinase and both AP-1 and NF-kappaB transcription factors. Contact lenses did not affect surface expression of Toll-like receptor-2, -4 or -5, but did block antigen activation of AP-1, but not NF-kappaB, transcription factors. These data show that contact lenses can interfere with epithelial defense responses to bacterial antigens in vitro, and if translated in vivo, could help predispose the cornea to infection.

New HIV-drug inhibits in vitro bladder cancer migration and invasion.

OBJECTIVE: The CXCR4/CXCL12 axis appears crucial in the metastasis of bladder cancer. Our aim was to evaluate the potency of the CXCR4 antagonist, 4F-benzoyl-TE14011 (4F-bTE), as an anti-metastatic drug in this disease. In this study, we assessed the ability of 4F-bTE to inhibit tumor cell motility, invasion through extracellular matrix (ECM), matrix metalloproteinase (MMP) secretion and cytoskeletal responses to chemokine. METHODS: To assess the degree to which cells could migrate and invade ECM under various conditions, we used TCCSUP bladder cancer cells in a Boyden chamber system. To monitor actin polymerization, we stained cells on chamber slides with AlexaFluor 594 phalloidin. To measure matrix-metalloproteinase-2 and -9 (MMP) activity, we used gelatin zymography. To assess the effects of the CXCR4 antagonist 4F-bTE on each of the above parameters, we exposed bladder cancer cells either to chemokine CXCL12, alone, or to both CXCL12 and 4F-bTE. We also monitored cells for apoptotic and necrotic changes during drug treatment. RESULTS: The CXCR4 antagonist 4F-bTE markedly decreased CXCL12-induced bladder cancer cell migration and ECM invasion in Boyden chamber assays. The antagonist also blocked chemokine-induced actin polymerization as well as the induction of MMP-2 and MMP-9 in these cells. CONCLUSION: The CXCR4 antagonist 4F-bTE has the potential to inhibit expression of the metastatic phenotype and may provide therapeutic value to patients.

Prolonged herbicide-induced vegetation changes in a regenerating boreal aspen clearcut.

A soil-active herbicide (hexazinone) was applied (0, 2, and 4 kg/ha of active ingredient) in a 3-year-old regenerating boreal Populus tremuloides Michx. (aspen) clearcut to determine its effect on the compositional and structural development of the vegetation. Woody stem densities and plant foliar cover were evaluated prior to and 2, 6, and 17 years after treatment. Herbicide treatment at the 2 and 4 kg/ha rates reduced tree and total woody stem densities relative to the 0 kg/ha level. The 4 kg/ha level reduced stem densities by 27% 17 years after treatment. The primary reductions occurred in Amelanchier alnifolia (Nutt.) Nutt. ex M. Roemer (saskatoon) and Rosa acicularis Lindl. (wild rose); whereas Corylus cornuta Marsh. (beaked hazelnut) and Viburnum edule (Michx.) Raf. (low-bush cranberry) stem densities increased. Notable herbicide-caused foliar cover reductions at the 4 kg/ha level occurred in Eurybia conspicua (Lindl.) Nesom. (showy aster), Mertensia paniculata (Ait.) G. Don. (tall mertensia), Rubus pubescens Raf. (dewberry), and Spiraea betulifolia Pallas (spiraea), but Aralia nudicaulis L. (sarsaparilla), Cornus canadensis L. (bunchberry), and Symphyotrichum ciliolatum (Lindl.) A.&D. Lve (Lindley's aster) increased. Less distinctive but similar changes occurred in the 2 kg/ha treatment. Total plant cover, species richness, and species dominance concentration were similar among treatments. Eight distinctive forest understory-types were recognized among treatments in Year 17. Between the 0 and 4 kg/ha treatments, five understory-types differed in their frequency of occurrence. Hexazinone did not improve the survival of silviculturally planted Picea glauca (Moench) Voss (white spruce) seedlings relative to untreated sites, but the 4 kg/ha treatment level did increase Pinus contorta Dougl. ex Loud. (lodgepole pine) survival from 12 to 34%. Surviving seedlings had significantly greater height and basal diameter growth than those at the 0 kg/ha sites, particularly the 4 kg/ha treatment.

Calcium mobilisation and CCK secretion induced by modified fatty acids and latex microspheres reveal dual receptor mechanisms for lipid stimulation of STC-1 cells.

How fatty acids stimulate enteroendocrine cells to release cholecystokinin (CCK) is largely unknown. Recently, we proposed that the murine enteroendocrine cell line, STC-1, responds to insoluble fatty acid aggregates rather than fatty acid monomers in solution. This hypothesis led to two testable predictions. First, other insoluble particles of similar size but unrelated to fatty acid may be able to stimulate STC-1 cells in a similar fashion to dodecanoic acid and second, fatty acid sensing in STC-1 cells should be fairly insensitive to chemical modifications of the fatty acid as long as these modifications do not greatly alter the ability of the molecule to form insoluble aggregates. We used several analogues of dodecanoic acid and several varieties of latex microsphere (varying in size and surface charge) to see whether the predictions of our model hold. We found that while there was at least one latex microsphere that could induce CCK secretion and calcium mobilisation in STC-1 cells, there was a very poor correlation between the presence of insoluble aggregates and a cellular response. Instead the most important property, determining the potency of fatty acid analogues as stimulants of CCK secretion, was their amphipathicity. Removal of either the polar head or lipophilic tail completely abolished the ability of a given fatty acid analogue to stimulate STC-1 cells. These data suggested that while fatty acids can stimulate cells as aggregates, they may also be acting in monomeric form with the oil:water partitioning coefficient playing a crucial role. We finally resolved this issue with the observation that the sulfate ion greatly altered the response of STC-1 cells to monomeric dodecanoic acid. In the presence of sulfate, STC-1 cells will only respond to dodecanoic acid aggregates whereas when sulfate is replaced with chloride the cells clearly respond to dodecanoic acid monomers which are completely in solution. In summary, we propose that dodecanoic acid can stimulate STC-1 cells via two separate pathways one involving fatty acid monomers in solution and one involving fatty acid aggregates. Which pathway dominates depends on the presence of sulfate in the extracellular medium.