Human monocytes augment invasiveness and proteolytic activity of inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer, and here, we examined in vitro the interactions between the human IBC cell line SUM149 and U937 human naive monocytes. We found an altered morphology, enhanced invasiveness and proteolytic activity of SUM149 cells when cultured with U937 cells or in U937-conditioned media (U937-CM). Increases in expression and activity of the cysteine protease cathepsin B and expression of caveolin-1 were also detected in SUM149 cells grown in U937-CM, thus suggesting a contribution of these proteins to the augmented invasion through and proteolysis of the extracellular matrix by the IBC cells.     

Two green and sensitive spectrofluorimetric approaches for determination of Ambroxol and guaifenesin in their single and combined pharmaceutical formulations

Ambroxol hydrochloride (AMX) and guaifenesin (GFN) are approved drugs utilized to treat coughs through their potent mucolytic and expectorant properties. Due to their massive, combined administration in many illnesses, there is a persistent need for their concurrent estimation in different pharmaceutical formulations. Two sensitive, environmentally friendly spectrofluorimetric methods were developed. AMX was determined using the first method (I) without interference from GFN. This method depends on the quenching of Erythrosine B (EB) native fluorescence at 552 nm after excitation at 527 nm due to the formation of a non-fluorescent AMX-EB ion-pair complex in Britton–Robinson buffer (BRB) solution pH (3.5). The concentration plot is linear over the 0.25–5.0 μg/mL range, with a mean percent found value of 99.74%. Method (II) depends on measuring the native fluorescence of aqueous GFN solution at two analytical wavelengths, either 300 or 600 nm, after excitation at 274 nm. Relative fluorescence intensity (RFI)–concentration plots are linear over the ranges of 0.02–0.5 and 0.1–2.0 μg/ml, with mean percent found at 99.96% and 99.91% at dual wavelengths, respectively. The proposed methods were successfully applied to assay both drugs in raw materials and different single and combined pharmaceutical formulations. These methods have been thoroughly validated following International Committee on Harmonisation (ICH) guidelines. National Environmental Methods Index, Analytical Eco-Scale, and Green Analytical Procedure Index were used to prove greenness, thereby enhancing their applicability. The proposed techniques provide straightforward, precise, and cost-effective solutions for routine formulation analysis in quality control laboratories.     

Insulin resistance and early virological response in chronic HCV infection

BackgroundStudies revealed that insulin resistance is associated with fibrosis progression and has negative impact on sustained virological response after standard antiviral therapy in patients with chronic hepatitis C (CHC).AimTo assess the role of IR on progression of liver fibrosis and early virological response (EVR) rates in patients with chronic hepatitis C infection.Patients and methodsThe study population comprised 79 subjects who underwent combination therapy for CHC. Laboratory investigations in the form of glucose, insulin, bilirubin, alanine aminotransferase (ALT), cholesterol and triglycerides and liver biopsy were done for all patients. Insulin resistance was calculated using the homeostasis model of IR (HOMA-IR).ResultsIR was increased (>2 IU) in 31 (40.7%) of patients. Early virological response was achieved among 37 patients (48.7%). No difference in EVR, viral load or grade of liver fibrosis between patients with and without IR. A significant positive correlation was found between IR and liver steatosis.ConclusionInsulin resistance is a common finding in CHC, it is associated with increase liver steatosis. However it has no impact on EVR to combined interferon ribavirin therapy, viral load or necroinflammation.     

Structural basis of calcineurin activation by calmodulin

Calcineurin is the only known calmodulin (CaM) activated protein phosphatase, which is involved in the regulation of numerous cellular and developmental processes and in calcium-dependent signal transduction. Although commonly assumed that CaM displaces the autoinhibitory domain (AID) blocking substrate access to its active site, the structural basis underlying activation remains elusive. We have created a fused ternary complex (CBA) by covalently linking three polypeptides: CaM, calcineurin regulatory B subunit (CnB) and calcineurin catalytic A subunit (CnA). CBA catalytic activity is comparable to that of fully activated native calcineurin in the presence of CaM. The crystal structure showed virtually no structural change in the active site and no evidence of CaM despite being covalently linked. The asymmetric unit contains four molecules; two parallel CBA pairs are packed in an antiparallel mode and the large cavities in crystal packing near the calcineurin active site would easily accommodate multiple positions of AID-bound CaM. Intriguingly, the conformation of the ordered segment of AID is not altered by CaM; thus, it is the disordered part of AID, which resumes a regular α-helical conformation upon binding to CaM, which is displaced by CaM for activation. We propose that the structural basis of calcineurin activation by CaM is through displacement of the disordered fragment of AID which otherwise impedes active site access.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

The expression of interleukin-8 and interleukin-8 receptors in endometrial carcinoma

Interleukin-8 (IL-8) is a pro-inflammatory cytokine which exerts its effects via binding to 2 receptors, CXCR1 and CXCR2 and is known to promote angiogenesis, mitogenesis and motogenesis in cancer. IL-8 is over expressed in endometrial carcinoma, but the expression of CXCR1 and CXCR2 in endometrial carcinoma has not been previously investigated. The aim of this study was to determine the expression of IL-8 receptors in endometrial carcinoma. The expression of CXCR1 and CXCR2 was studied in endometrial carcinomas and normal endometrium by immunohistochemistry in 101 tumours. IL-8 and IL-8 receptor expression was also studied by Real-time quantitative PCR (RT-qPCR) in 17 tumours in comparison to normal endometrium. The expression profile was correlated to the clinico-pathological features of the tumours. Immunohistochemistry showed CXCR1 and CXCR2 were expressed in all cases of endometrial carcinoma, with CXCR1 showing stronger expression. There was a statistically significant correlation between CXCR2 staining intensity and tumour grade (P=0.012) and disease free survival (P=0.015) independently. On RT-qPCR, 14/17, 15/17 and 16/17 tumours showed significant increase in IL-8, CXCR1 and CXCR2 expression levels in comparison to normal endometrium, with median fold increase of 42-fold, 51-fold and 27-fold, respectively. This is the first report of the expression of IL-8 receptors in endometrial carcinoma and the results show an association between IL-8 and IL-8 receptors and the pathogenesis of endometrial carcinoma, and represent potential prognostic biomarkers and therapeutic targets.     

Phytoplankton and zooplankton as indicators of water quality in Discovery Bay,Jamaica

Several investigations exist which use planktonic communities as indicators of water quality in Jamaican and Caribbean Bays, however, few are conducted before there are obvious effects of eutrophication. Therefore, most of our ‘baseline’ data are for bays already severely affected by pollution. This study was conducted to assess water quality in Discovery Bay, Jamaica, before there were severe signs of eutrophication. The bay was monitored over a 12-month period (October 1995–September 1996) using 10 stations. Physicochemical data indicated a well mixed upper 5 m of water column, below which discontinuities in temperature/salinity profiles indicated the influence of colder, more saline waters associated with deep offshore currents. Physicochemical variables were within the range for oligotrophic systems with a tendency towards mesotrophic in localized areas close to the shoreline. Signs of anthropogenic stress were associated with the eastern, southwestern and western sections of the bay. Of the over 120 species of phytoplankton found in the waters of Discovery Bay, most were neritic/oceanic and diatoms dominated while 11 were found to be potentially harmful species. While these harmful species occurred at all stations they occurred most frequently at stations on the eastern side of the bay. About 107 zooplankton species were identified, 52 of which were copepods. The species also represented a mix of neritic and oceanic taxa and mean abundances for the area ranged from 1077 m⁻³ at the mouth of the bay to 3794 m⁻³ close to the south shore (station 6). Generally stations closest to shore had greater zooplankton abundances than centrally located bay stations and stations close to oceanic influence. Acartia tonsa and Lucifer faxoni showed greatest densities at shoreline areas of the bay while Oithona plumifera, Undinula vulgaris and Temora stylifera were important at stations closest to oceanic influences. These species were thus considered as indicators of these different areas within the bay. From physicochemical data and the planktonic assemblage, Discovery Bay cannot be considered polluted, it is still more accurately classified as generally pristine with mesotrophic zones in the eastern and southeastern sections of the bay. These data therefore provide a real baseline of conditions for similar tropical coastal embayments.     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.