An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors.

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10-/- mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.

The novel compound OSI-461 induces apoptosis and growth arrest in human acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy. Treatment of patients suffering from high-risk AML as defined by clinical parameters, cytogenetics, and/or molecular analyses is often unsuccessful. OSI-461 is a pro-apoptotic compound that has been proposed as a novel therapeutic option for patients suffering from solid tumors like prostate or colorectal carcinoma. But little is known about its anti-proliferative potential in AML. Hence, we treated bone marrow derived CD34(+) selected blast cells from 20 AML patients and the five AML cell lines KG-1a, THP-1, HL-60, U-937, and MV4-11 with the physiologically achievable concentration of 1 µM OSI-461 or equal amounts of DMSO as a control. Following incubation with OSI-461, we found a consistent induction of apoptosis and an accumulation of cells in the G2/M phase of the cell cycle. In addition, we demonstrate that the OSI-461 mediated anti-proliferative effects observed in AML are associated with the induction of the pro-apoptotic cytokine mda-7/IL-24 and activation of the growth arrest and DNA-damage inducible genes (GADD) 45α and 45γ. Furthermore, OSI-461 treated leukemia cells did not regain their proliferative potential for up to 8 days after cessation of treatment following the initial 48 h treatment period with 1 µM OSI-461. This indicates sufficient targeting of the leukemia-initiating cells in our in vitro experiments through OSI-461. The AML samples tested in this study included samples from patients who were resistant to conventional chemotherapy and/or had FLT3-ITD mutations demonstrating the high potential of OSI-461 in human AML.

Clinical profile of severe Plasmodium vivax malaria in a tertiary care centre in Mumbai from June 2010-January 2011.

BACKGROUND: Plasmodium vivax is the most widely distributed human malaria parasite with an at risk population of 2.5 billion persons. With the implementation of molecular diagnosis, it has become evident that P. vivax monoinfection could also result in multiple organ dysfunction and severe life-threatening disease as seen in P. falciparum infection. AIMS AND OBJECTIVES: To note the clinical profile of patients with severe vivax malaria with regards to demographic, clinical and biochemical profile and its outcome. To compare the profile of falciparum malaria with vivax malaria. METHOD AND MATERIAL: We recruited 711 patients fulfilling the criteria for severe malaria during the study period from June 2010 to Jan 2011. Detailed history and examination findings were noted in all the patients. All the patients were subjected to routine haematological and biochemical investigations. The end points were discharge from wards or death due to malaria. RESULTS: We had 711 patients with severe malaria of which 488 (68.53%) patients had severe vivax and 223 (31.32%) had severe falciparum malaria. Amongst vivax group, 351 (71.92%) were males and 137 (28.07%) females. Thrombocytopenia (89.13%) was the most common complication followed by renal (31.96%), hepatic (19.46%) cerebral (8.19%) and pulmonary (1.63%) involvement. Most patients were in the age group of 21-30 years and mortality increased with increasing age. The mortality observed in severe vivax malaria was 9.01% (44/488), as compared to falciparum malaria where it was 16.14% (80/223). CONCLUSIONS: Severe vivax malaria is now very common with increasing mortality. The mortality in vivax malaria increases with increasing age. Thrombocytopenia is very common in severe vivax infection. Also, renal, hepatic, lung and cerebral involvement are also occur with increasing frequency. Along with age, severe metabolic acidosis is an independent risk factor for fatal outcome.

Polydopamine and gelatin coating for rapid endothelialization of vascular scaffolds.

Rapid endothelialization helps overcome the limitations of small-diameter vascular grafts. To develop biomimetic non-thrombogenic coatings supporting endothelialization, medical-grade polyurethane (PU) nanofibrous mats and tubular scaffolds with a diameter below 6 mm prepared by solution blow spinning were coated with polydopamine (PDA), or PDA and gelatin (PDA/Gel). The scaffolds were characterized by scanning electron microscopy, porosity measurement, tensile testing, wettability, Fourier Transform Infrared spectroscopy, and termogravimetric analysis, followed by the measurement of coating stability on the tubular scaffolds. The effect of coating on scaffold endothelialization and hemocompatibility was evaluated using human umbilical vein endothelial cells (HUVECs) and human platelets, showing low numbers of adhering platelets and significantly higher numbers of HUVECs on PDA- and PDA/Gel-coated mats compared to control samples. Tubular PU scaffolds and commercial ePTFE prostheses coated with PDA or PDA/Gel were colonized with HUVECs using radial magnetic cell seeding. PDA/Gel-coated samples achieved full endothelial coverage within 1-3 days post-endothelialization. Altogether, PDA and PDA/Gel coating significantly enhance the endothelialization on the flat surfaces, tubular small-diameter scaffolds, and commercial vascular prostheses. The presented approach constitutes a fast and efficient method of improving scaffold colonization with endothelial cells, expected to work equally well upon implantation.

The non-steroidal anti-inflammatory drugs Sulindac sulfide and Diclofenac induce apoptosis and differentiation in human acute myeloid leukemia cells through an AP-1 dependent pathway.

Acute myeloid leukemia is a heterogeneous disease with varying genetic and molecular pathologies. Non-steroidal anti-inflammatory drugs (NSAIDs) have been proven to possess significant anti-proliferative potential in various cancer cells in vitro and in vivo. Hence, treatment with these agents can be utilized to study disease specific anti-proliferative pathways. In this study, a total number of 42 bone marrow derived CD34(+) selected de novo AML patient samples and the AML cell lines THP-1 and HL-60 were treated with the NSAIDs Sulindac sulfide and Diclofenac. We analyzed viability, apoptosis, differentiation and addressed the molecular mechanisms involved. We found a consistent induction of apoptosis and to some extent an increased myeloid differentiation capacity in NSAID treated AML cells. Comprehensive protein and gene expression profiling of Diclofenac treated AML cells revealed transcriptional activation of GADD45α and its downstream MAPK/JNK pathway as well as increased protein levels of the caspase-3 precursor. This pointed towards a role of the c-Jun NH(2)-terminal kinase (JNK) in NSAID mediated apoptosis that we found indeed to be dependent on JNK activity as addition of a specific JNK-inhibitor abrogated apoptosis. Furthermore, the AP-1 transcription factor family members' c-Jun, JunB and Fra-2 were transcriptionally activated in NSAID treated AML cells and re-expression of these transcription factors led to activation of GADD45α with induction of apoptosis. Mechanistically, we demonstrate that NSAIDs induce apoptosis in AML through a novel pathway involving increased expression of AP-1 heterodimers, which by itself is sufficient to induce GADD45α expression with consecutive activation of JNK and induction of apoptosis.

Study of patient characteristics/profile and factors determining the (immediate) outcome in spontaneous subarachnoid hemorrhage.

AIMS AND OBJECTIVES: The aims of the study were to delineate the risk factors, type and location of underlying pathology, outcome and the determinants of outcome in patients with spontaneous subarachnoid hemorrhage. MATERIAL AND METHODS: Forty consecutive patients with spontaneous subarachnoid hemorrhage on neuroimaging scan were recruited over a period of 1 year. Risk factors profile, site and type of pathology seen on angiography were assessed in all patients. Patient outcome at the end of hospital stay was assessed using the Glasgow Outcome Scale (GOS) and was analysed with respect to demographic factors, premorbid risk factors, initial WFNS scoring, neuroimaging findings and complications during in-hospital stay. RESULTS: Of the total 40 patients in the study, 43% were males and 57% were females. The mean age in study group was 49.63 yrs (SD 13.12). Fifty percent patients were hypertensive, 22.5% were smokers and alcohol intake was reported by 17.5%. Saccular aneurysms were seen in 80% patients and arteriovenous malformations in 7.5%. Aneurysms were more common in the anterior circulation than in the posterior circulation. Poor outcome was associated with higher age, hyponatremia, higher World Federation of Neurosurgeons (WFNS) grade on admission, presence of vasospasm on angiography, fever any time during the course in hospital and requirement of ventilatory support. CONCLUSION: Gender, site of aneurysmal bleeding (anterior/posterior circulation) and procedure performed (coiling/clipping) do not influence the immediate outcome of patients with subarachnoid hemorrhage. Further studies on Indian subset of patients are necessary to determine the patient characteristics and factors influencing the long-term outcome in spontaneous subarachnoid hemorrhage.

Regulation of CEACAM Family Members by IBD-Associated Triggers in Intestinal Epithelial Cells, Their Correlation to Inflammation and Relevance to IBD Pathogenesis.

Carcinoembryogenic antigen cellular adhesion molecules (CEACAMs) are intercellular adhesion molecules highly expressed in intestinal epithelial cells. CEACAM1, -3, -5, -6, -7 are altered in patients suffering from colon cancer and inflammatory bowel diseases (IBD), but their role in the onset and pathogenesis of IBD is not well known. Herein, we aim to correlate CEACAM1, -3, -5, -6, -7 expression to the degree of inflammation in pediatric and adult IBD colon biopsies and to examine the regulation of CEACAMs on human intestinal epithelial cell lines (C2BBe1/HT29) by different IBD-associated triggers (cytokines, bacteria/metabolites, emulsifiers) and IBD-drugs (6-Mercaptopurine, Prednisolone, Tofacitinib). Biopsies from patients with pediatric Crohn's disease (CD) and adult ulcerative colitis (UC, active/inactive disease) showed a significant increase in CEACAM3, -5, -6 expression, while CEACAM5 expression was reduced in adult CD patients (active/inactive disease). Intestinal epithelial cells cultured with a pro-inflammatory cytokine cocktail and Adherent-invasive Escherichia coli (AIEC) showed a rapid induction of CEACAM1, -5, -7 followed by a reduced RNA and protein expression overtime and a constant expression of CEACAM3, correlating with IL-8 expression. Cells cultured with the emulsifier polysorbate-80 resulted in a significant induction of CEACAM3, -5, -6, -7 at a late time point, while SCFA treatment reduced CEACAM1, -5, -7 expression. No major alterations in expression of CEACAMs were noted on cells cultured with the commensal Escherichia coli K12 or the pathogen Salmonella typhimurium. IBD drugs, particularly Tofacitinib, significantly reduced cytokine-induced CEACAM1, -3, -5, -6, -7 expression associated with a reduced IL-8 secretion. In conclusion, we provide new evidence on the regulation of CEACAMs by different IBD-associated triggers, identifying a role of CEACAMs in IBD pathogenesis.

Low RPS14 expression is common in myelodysplastic syndromes without 5q- aberration and defines a subgroup of patients with prolonged survival.

To further clarify the role of ribosomal protein S14 (RPS14) in myelodysplastic syndrome, we examined RPS14 transcription in bone marrow derived CD34+ cells from patients with non-5q- myelodysplastic syndrome and found a reduced expression of RPS14 in 51 of 72 (71%) patients. MDS patients with an intermediate-1 risk (INT-1) score according to the international prognostic scoring system and low RPS14 expression had a superior median overall survival of not reached versus 25 months compared to INT-1 patients with high RPS14 expression (p=0.0249). Using multivariate analysis, the RPS14 expression status was confirmed as an independent predictor for survival in INT-1 patients.

Emerging applications of upconverting nanoparticles in intestinal infection and colorectal cancer.

Colorectal cancer is the abnormal growth of cells in colon or rectum. Recent findings have acknowledged the role of bacterial infection and chronic inflammation in colorectal cancer initiation and progression. In order to detect and treat precancerous lesions, new tools are required, which may help to prevent or identify colorectal cancer at an early stage. To date, several different screening tests are available, including endoscopy, stool-based blood tests, and radiology-based tests. However, these analyses either lack sensitivity or are of an invasive nature. The use of fluorescently labeled probes can increase the detection sensitivity. However, autofluorescence, photobleaching, and photodamage are commonly encountered problems with fluorescence imaging. Upconverting nanoparticles (UCNPs) are recently developed lanthanide-doped nanocrystals that can be used as light-triggered luminescent probes and in drug delivery systems. In this review, we comprehensively summarize the recent developments and address future prospects of UCNP-based applications for diagnostics and therapeutic approaches associated with intestinal infection and colorectal cancer.

Carcinoembryonic antigen (CEACAM) family members and Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), is a chronic intestinal inflammatory condition with increasing incidence worldwide and whose pathogenesis remains largely unknown. The collected evidence indicates that genetic, environmental and microbial factors and a dysregulated immune response are responsible for the disease. IBD has an early onset and long term sufferers present a higher risk of developing colitis associated cancer (CAC). The carcinoembryonic antigen-related adhesion molecules (CEACAM) are a subgroup of the CEA family, found in a range of different cell types and organs including epithelial cells in the intestine. They can act as intercellular adhesions molecules for e.g. bacteria and soluble antigens. CEACAMs are involved in a number of different processes including cell adhesion, proliferation, differentiation and tumour suppression. Some CEACAMs such as CEACAM1, CEACAM5 and CEACAM6 are highly associated with cancer and are even recognised as valid clinical markers for certain cancer forms. However, their role in IBD pathogenesis is less understood. The purpose of this review is to provide a comprehensive summary of published literature on CEACAMs and intestinal inflammation (IBD). The interactions between CEACAMs and bacteria adhesion in relation to IBD pathophysiology will be addressed and potential new therapeutic and diagnostic opportunities will be identified.

Easy-to-Prepare Coating of Standard Cell Culture Dishes for Cell-Sheet Engineering Using Aqueous Solutions of Poly(2-n-propyl-oxazoline).

Cell-sheet technology is a well-known method by which cells are grown on thermoswitchable substrates that become nonadhesive upon cooling, such that a complete layer of adherent cells, along with the produced extracellular matrix, detaches as a sheet. Polymers that exhibit a lower critical solution temperature (LCST) below physiological temperature in water, commonly poly(N-isopropylacrylamide) (PNIPAM), are covalently grafted or, for block copolymers, physisorbed onto substrates in a monomolecular thin film to achieve this. Consequently, such substrates, and the polymers required for film formation, can only be prepared in a chemical lab with profound macromolecular expertise. In this study, we present an easy and robust method to coat standard cell culture dishes with aqueous solutions of commercially available poly(2-n-propyl-2-oxazoline) (PnPrOx), a polymer that exhibits LCST behavior. Different standard cell culture dishes were repeatedly coated with 0.1 wt % aqueous solutions of PnPrOx and dried in an oven to create a fully covered and thermoresponsive surface. Using this PnPrOx surface a variety of cell types including endothelial cells, mesenchymal stem cells, and fibroblasts, were seeded and cultured until confluency. By decreasing the temperature to 16 °C, viable cell sheets were detached within cell-type dependent time frames and could be harvested for biological analysis. We show that the cytoskeleton rearranges, leading to a more contracted morphology of the cells in the detached cell sheet. The cellular junctions between single cells within the sheet could be detected using immunostainings, indicating that strong and intact intracellular contacts are preserved in the harvested sheets.

Pegylated granulocyte colony-stimulating factor mobilizes CD34+ cells with different stem and progenitor subsets and distinct functional properties in comparison with unconjugated granulocyte colony-stimulating factor.

BACKGROUND: Pegylated granulocyte colony-stimulating factor (G-CSF) has recently been introduced as a new compound for mobilization of CD34(+) hematopoietic stem and progenitor cells. In this study, we compared the molecular and functional characteristics of CD34(+) cells mobilized by pegylated G-CSF with those mobilized by unconjugated G-CSF. DESIGN AND METHODS: Gene expression of immunomagnetically enriched CD34(+) cells from leukapheresis products of patients who were given pegylated-G-CSF or unconjugated G-CSF was analyzed using Affymetrix HG Focus microarrays and quantitative reverse transcriptase polymerase chain reaction. Flow cytometry and fluorescence activated cell sorting was conducted to assess the CD34(+) subset composition and to obtain Lin(-), CD34(+), CD38(-) hematopoietic stem cells. Cell cycle assays and clonogenic assays were performed for functional corroboration. RESULTS: Pegylated G-CSF and unconjugated G-CSF mobilized CD34(+) and hematopoietic stem cells with different molecular phenotypes and functional properties. The CD34(+) cells mobilized by pegylated G-CSF had higher expression levels of genes indicative of early hematopoiesis, including HOXA9, MEIS1 and GATA3. We found lower expression of genes characteristic of erythroid and later stages of myeloid differentiation and a lower functional burst-forming unit erythroid/colony-forming unit-granulocyte-macrophage ratio. Consistently, greater numbers of hematopoietic stem cells and common myeloid progenitors and fewer megakaryocyte-erthrocyte progenitors were found in the pegylated-G-CSF-mobilized CD34(+) cells. Additionally, sorted pegylated-G-CSF-mobilized hematopoietic stem cells displayed higher expression of HOXA9 in comparison to G-CSF-mobilized hematopoietic stem cells. In line with the gene expression data, CD34(+) cells mobilized by pegylated G-CSF, as well as sorted hematopoietic stem cells, showed a significantly greater cell cycle activity. CONCLUSIONS: Stimulation with pegylated-G-CSF or G-CSF results in different expression of key regulatory genes and different functional properties of mobilized hematopoietic stem cells as well as their progeny, a finding that might be relevant for the application of these cells in blood stem cell transplantation.

Tumefactive demyelination.

We present a case of 22 year old female who had pulmonary tuberculosis followed by tuberculous meningitis and tuberculomas in past. This time she presented to us with right hemiparesis and altered sensorium. Diagnosis of tumefactive demyelination was made on the basis of typical MRI findings. Patient showed good response to steroids.

Cardiac amyloidosis.

We present a 54 year old male who presented with congestive cardiac failure and was diagnosed as restrictive cardiomyopathy with mild mitral regurgitation on 2D echocardiography. Cardiac amyloidosis was diagnosed in view of renal biopsy revealing amyloid deposition. Patient did not have any obvious etiology for secondary amyloidosis.

Soy Protein-Based Composite Hydrogels: Physico-Chemical Characterization and In Vitro Cytocompatibility.

Novel composite hydrogels based on the combination of alginate (Alg), soy protein isolate (SPI) and bioactive glass (BG) nanoparticles were developed for soft tissue engineering. Human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts were cultivated on hydrogels for 7, 14 and 21 days. Cell morphology was visualized using fluorescent staining at Days 7 and 14 for fibroblast cells and Days 14 and 21 for HUVEC. Metabolic activity of cells was analyzed using a colorimetric assay (water soluble tetrazolium (WST) assay). Compared to pure Alg, Alg/SPI and Alg/SPI/BG provided superior surfaces for both types of cells, supporting their attachment, growth, spreading and metabolic activity. Fibroblasts showed better colonization and growth on Alg/SPI/BG hydrogels compared to Alg/SPI hydrogels. The results indicate that such novel composite hydrogels might find applications in soft tissue regeneration.

Hydrogel matrices based on elastin and alginate for tissue engineering applications.

Hydrogels from natural polymers are widely used in tissue engineering due to their unique properties, especially when regarding the cell environment and their morphological similarity to the extracellular matrix (ECM) of native tissues. In this study, we describe the production and characterization of novel hybrid hydrogels composed of alginate blended with elastin from bovine neck ligament. The properties of elastin as a component of the native ECM were combined with the excellent chemical and mechanical stability as well as biocompatibility of alginate to produce two hybrid hydrogels geometries, namely 2D films obtained using sonication treatment and 3D microcapsules produced by pressure-driven extrusion. The resulting blend hydrogels were submitted to an extensive physico-chemical characterization. Furthermore, the biological compatibility of these materials was assessed using normal human dermal fibroblasts, indicating the suitability of this blend for soft tissue engineering.

Cell specificity of magnetic cell seeding approach to hydrogel colonization.

Tissue-engineered scaffolds require an effective colonization with cells. Superparamagnetic iron oxide nanoparticles (SPIONs) can enhance cell adhesion to matrices by magnetic cell seeding. We investigated the possibility of improving cell attachment and growth on different alginate-based hydrogels using fibroblasts and endothelial cells (ECs) loaded with SPIONs. Hydrogels containing pure alginate (Alg), alginate dialdehyde crosslinked with gelatin (ADA-G) and Alg blended with G or silk fibroin (SF) were prepared. Endothelial cells and fibroblasts loaded with SPIONs were seeded and grown on hydrogels for up to 7 days, in the presence of magnetic field during the first 24 h. Cell morphology (fluorescent staining) and metabolic activity (WST-8 assay) of magnetically-seeded versus conventionally seeded cells were compared. Magnetic seeding of ECs improved their initial attachment and further growth on Alg/G hydrogel surfaces. However, we did not achieve an efficient and stable colonization of ADA-G films with ECs even with magnetic cell seeding. Fibroblast showed good initial colonization and growth on ADA-G and on Alg/SF. This effect was further significantly enhanced by magnetic cell seeding. On pure Alg, initial attachment and spreading of magnetically-seeded cells was dramatically improved compared to conventionally-seeded cells, but the effect was transient and diminished gradually with the cessation of magnetic force. Our results demonstrate that magnetic seeding improves the strength and uniformity of initial cell attachment to hydrogel surface in cell-specific manner, which may play a decisive role for the outcome in tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2948-2956, 2017.

Effect of cortical cleaving hydrodissection on posterior capsule opacification in age-related nuclear cataract.

PURPOSE: To evaluate the effect of multiquadrant cortical cleaving hydrodissection on the development of posterior capsule opacification (PCO) after phacoemulsification. SETTING: Iladevi Cataract and IOL Research Center, Ahmedabad, India. METHODS: A prospective randomized triple blind clinical trial was conducted in 86 eyes (86 patients) having phacoemulsification. Eyes were assigned randomly to Group 1, multiquadrant cortical cleaving hydrodissection and hydrodelineation (n = 48 eyes), or Group 2, no multiquadrant cortical cleaving hydrodissection, only hydrodelineation (n = 38 eyes). Age-related nuclear sclerosis (grading system of 1 to 5) and age greater than 50 years were included. Diabetes mellitus and associated eye diseases were excluded. Standard phacoemulsification with implantation of an AcrySof MA30BA intraocular lens (IOL) was done in both groups. High-resolution digital retroillumination images of the posterior capsule were analyzed at 4 years using POCOman software. Seven patients dropped out (8.1%). Outcome measures were incidence and percentage area of PCO. Test of proportions, independent sample t test, and 95% confidence intervals (CIs) were noted. RESULTS: In Groups 1 and 2, the mean age was 58 years +/- 4 (SD) and 57 +/- 5 years, respectively (P = .702; 95% CI, 1.90-2.81); mean follow-up was 48.4 +/- 2.5 months and 49.3 +/- 1.9 months, respectively (P = .687; 95% CI, 1.186-0.7856); incidence of PCO was 11 of 44 (25%) and 12 of 35 (34.3%), respectively (P = .317; 95% CI, 0.359-0.0889); percentage area of PCO was 14.3% and 25.6%, respectively (P = .006; 95% CI, -19.19 to -3.44). One of 44 eyes (2.27%) and 2 of 35 eyes (5.7%) in the groups, respectively, had a neodymium:YAG capsulotomy. CONCLUSION: Although no difference was noted in the incidence of PCO, in eyes that had PCO, the percentage of the area of the central posterior capsule involved by PCO was significantly lower in eyes that had multiquadrant cortical cleaving hydrodissection than in those that did not.