Neutrophils as one of the major haptoglobin sources in mastitis affected milk

[[abstract]]The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to de. ne the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: 500 x 10(3) (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were similar to 10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immuno fluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation

A unique tetrameric structure of deer plasma haptoglobin - an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure

[[abstract]]Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by alpha 2-beta subunits or (alpha 2-beta)(n) (where n >= 3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the alpha-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (alpha-beta)(4), although the locations of -SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 M urea under reducing conditions (143 mM beta-mercaptoethanol), followed by renaturation, sustained the formation of (alpha-beta)(4), suggesting that the Hp tetramers are not randomly assembled. Interestingly, an alpha-chain monoclonal antibody (W1), known to recognize both human and deer alpha-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer alpha-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage

Genomic diversity of citrate fermentation in Klebsiella pneumoniae

<p>Abstract</p> <p>Background</p> <p>It has long been recognized that <it>Klebsiella pneumoniae </it>can grow anaerobically on citrate. Genes responsible for citrate fermentation of <it>K. pneumoniae </it>were known to be located in a 13-kb gene cluster on the chromosome. By whole genome comparison of the available <it>K. pneumoniae </it>sequences (MGH 78578, 342, and NTUH-K2044), however, we discovered that the fermentation gene cluster was present in MGH 78578 and 342, but absent in NTUH-K2044. In the present study, the previously unknown genome diversity of citrate fermentation among <it>K. pneumoniae </it>clinical isolates was investigated.</p> <p>Results</p> <p>Using a genomic microarray containing probe sequences from multiple <it>K. pneumoniae </it>strains, we investigated genetic diversity among <it>K. pneumoniae </it>clinical isolates and found that a genomic region containing the citrate fermentation genes was not universally present in all strains. We confirmed by PCR analysis that the gene cluster was detectable in about half of the strains tested. To demonstrate the metabolic function of the genomic region, anaerobic growth of <it>K. pneumoniae </it>in artificial urine medium (AUM) was examined for ten strains with different clinical histories and genomic backgrounds, and the citrate fermentation potential was found correlated with the genomic region. PCR detection of the genomic region yielded high positive rates among a variety of clinical isolates collected from urine, blood, wound infection, and pneumonia. Conserved genetic organizations in the vicinity of the citrate fermentation gene clusters among <it>K. pneumoniae</it>, <it>Salmonella enterica</it>, and <it>Escherichia coli </it>suggest that the13-kb genomic region were not independently acquired.</p> <p>Conclusion</p> <p>Not all, but nearly half of the <it>K. pneumoniae </it>clinical isolates carry the genes responsible for anaerobic growth on citrate. Genomic variation of citrate fermentation genes in <it>K. pneumoniae </it>may contribute to metabolic diversity and adaptation to variable nutrient conditions in different environments.</p

Excision of sympathetic ganglia and the rami communicantes with histological confirmation offers better early and late outcomes in Video assisted thoracoscopic sympathectomy

<p>Abstract</p> <p>Background</p> <p>Video-Assisted Thoracoscopic Sympathectomy (VATS) is an established minimally invasive procedure for thoracic sympathetic blockade in patients with hyperhidrosis, facial flushing and intractable angina. Various techniques using clips, diathermy and excision are used to perform sympathectomy. We present our technique of excision of the sympathetic chain with histological proof and the analysis of the early and late outcomes.</p> <p>Methods</p> <p>We evaluated 200 procedures in 100 consecutive patients, who underwent Video Assisted Thoracoscopic Sympathectomy by a single surgeon in our centre between September 1996 to March 2007. All patients had maximum medical therapy prior to surgery and were divided into 3 groups based on indications, Group 1(hyperhidrosis: 48 patients), Group 2 (facial flushing: 26 patients) and Group 3(intractable angina: 26 patients). The demography and severity of symptoms for each group were analysed. The endpoints were success rate, 30 day mortality, complications and patient's satisfaction.</p> <p>Results</p> <p>99 patients had bilateral VATS sympathectomy and 1 had unilateral sympathectomy. The conversion rate to open was 1(1%). All patients had successful removal of ganglia proven histologically with no perioperative mortality in our series. The complications included pneumothorax (5%), acute coronary syndrome (2%), transient Horner's syndrome (1%), transient paraesthesia (1%), wound infection (4%), compensatory hyperhidrosis (18%), residual flushing (3%) and wound pain (5%). There were five late deaths in the intractable angina group at a mean follow up of 36.7 months. Overall success rates of abolishing the symptoms were 96.3%, 87.5% and 95.2% for Group 1, 2 and 3 respectively.</p> <p>Conclusion</p> <p>Excision of the sympathetic chain with histological confirmation during VATS sympathectomy is a safe and effective method in treating hyperhidrosis, facial flushing and intractable angina with good long term results and satisfaction.</p

Calcium Homeostasis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Rationale: Cardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling. Objective: We investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs). Methods and Results: We differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs. Conclusions: The results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts. © 2011 The Author(s).published_or_final_versionSpringer Open Choice, 21 Feb 201

Bayesian estimation of genomic copy number with single nucleotide polymorphism genotyping arrays

<p>Abstract</p> <p>Background</p> <p>The identification of copy number aberration in the human genome is an important area in cancer research. We develop a model for determining genomic copy numbers using high-density single nucleotide polymorphism genotyping microarrays. The method is based on a Bayesian spatial normal mixture model with an unknown number of components corresponding to true copy numbers. A reversible jump Markov chain Monte Carlo algorithm is used to implement the model and perform posterior inference.</p> <p>Results</p> <p>The performance of the algorithm is examined on both simulated and real cancer data, and it is compared with the popular CNAG algorithm for copy number detection.</p> <p>Conclusions</p> <p>We demonstrate that our Bayesian mixture model performs at least as well as the hidden Markov model based CNAG algorithm and in certain cases does better. One of the added advantages of our method is the flexibility of modeling normal cell contamination in tumor samples.</p

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial

IMPORTANCE: Secretory phospholipase A2(sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measurewas a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95%CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95%CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246. Copyright 2014 American Medical Association. All rights reserved

The Evolution of Compact Binary Star Systems

We review the formation and evolution of compact binary stars consisting of white dwarfs (WDs), neutron stars (NSs), and black holes (BHs). Binary NSs and BHs are thought to be the primary astrophysical sources of gravitational waves (GWs) within the frequency band of ground-based detectors, while compact binaries of WDs are important sources of GWs at lower frequencies to be covered by space interferometers (LISA). Major uncertainties in the current understanding of properties of NSs and BHs most relevant to the GW studies are discussed, including the treatment of the natal kicks which compact stellar remnants acquire during the core collapse of massive stars and the common envelope phase of binary evolution. We discuss the coalescence rates of binary NSs and BHs and prospects for their detections, the formation and evolution of binary WDs and their observational manifestations. Special attention is given to AM CVn-stars -- compact binaries in which the Roche lobe is filled by another WD or a low-mass partially degenerate helium-star, as these stars are thought to be the best LISA verification binary GW sources.Comment: 105 pages, 18 figure

Expression of CD3-ζ on T-cells in primary cervical carcinoma and in metastasis-positive and -negative pelvic lymph nodes

Lymphocytic infiltrate is often present in cervical cancer lesions, possibly reflecting an ongoing, but ineffective, immune response to the tumour. Recently, evidence has accumulated for systemically impaired T-cell functions in cancer patients, associated with decreased expression of signal-transducing zeta (ζ) chain dimer molecules on circulating T-cells and NK-cells. Here, we report on the intralesional down-regulation of ζ chain expression on T-cells in cervical carcinoma. Paraffin-embedded or snap-frozen sections from 24 different cervical cancer specimens were studied. Paraffin-embedded tumour-positive (n = 7) and tumour-negative (n = 15) pelvic lymph nodes were also included in the study. Immunostaining was performed on consecutive sections with antibodies specific for CD3-ɛ or the CD3-associated ζ chain dimer. Antigen retrieval by sodium citrate/microwave treatment was essential for ζ staining of paraffin sections. The amount of ζ positive cells was quantitated and related to the number of CD3-ɛ+ cells in corresponding tumour areas. Of the 24 cervical cancer specimens studied, ζ chain dimer expression was reduced in seven cases and strongly reduced in the other 17 samples. In tonsil control sections, CD3-ɛ and CD3-ζ were always co-expressed in almost equal numbers. Also, both tumour-negative and -positive lymph nodes showed ζ chain expression which equalled that of CD3-ɛ expression. These data indicate that a decreased expression of signal-transducing ζ molecules on tumour-infiltrating T-cells is frequent in cervical cancer. The apparently unimpaired ζ chain expression within draining lymph nodes suggests that local tumour-derived factors at the primary site are instrumental in ζ chain down-regulation. © 1999 Cancer Research Campaig

Anticancer activity of a sub-fraction of dichloromethane extract of Strobilanthes crispus on human breast and prostate cancer cells in vitro

<p>Abstract</p> <p>Background</p> <p>The leaves of <it>Strobilanthes crispus </it>(<it>S. crispus</it>) which is native to the regions of Madagascar to the Malay Archipelago, are used in folk medicine for their antidiabetic, diuretic, anticancer and blood pressure lowering properties. Crude extracts of this plant have been found to be cytotoxic to human cancer cell lines and protective against chemically-induced hepatocarcinogenesis in rats. In this study, the cytotoxicity of various sub-fractions of dichloromethane extract isolated from the leaves of <it>S. crispus </it>was determined and the anticancer activity of one of the bioactive sub-fractions, SC/D-F9, was further analysed in breast and prostate cancer cell lines.</p> <p>Methods</p> <p>The dichloromethane extract of <it>S. crispus </it>was chromatographed on silica gel by flash column chromatography. The ability of the various sub-fractions obtained to induce cell death of MCF-7, MDA-MB-231, PC-3 and DU-145 cell lines was determined using the LDH assay. The dose-response effect and the EC₅₀values of the active sub-fraction, SC/D-F9, were determined. Apoptosis was detected using Annexin V antibody and propidium iodide staining and analysed by fluorescence microscopy and flow cytometry, while caspase 3/7 activity was detected using FLICA caspase inhibitor and analysed by fluorescence microscopy.</p> <p>Results</p> <p>Selected sub-fractions of the dichloromethane extract induced death of MCF-7, MDA-MB-231, PC-3 and DU-145 cells. The sub-fraction SC/D-F9, consistently killed breast and prostate cancer cell lines with low EC₅₀values but is non-cytotoxic to the normal breast epithelial cell line, MCF-10A. SC/D-F9 displayed relatively higher cytotoxicity compared to tamoxifen, paclitaxel, docetaxel and doxorubicin. Cell death induced by SC/D-F9 occurred via apoptosis with the involvement of caspase 3 and/or 7.</p> <p>Conclusions</p> <p>A dichloromethane sub-fraction of <it>S. crispus </it>displayed potent anticancer activities <it>in vitro </it>that can be further exploited for the development of a potential therapeutic anticancer agent.</p