A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Background

Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.

Methods and Findings

An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).

Conclusions

We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.     

Agonist versus antagonist muscle fatigue effects on thigh muscle activity and vertical ground reaction during drop landing

BackgroundAgonist and antagonist co-activation plays an important role for stabilizing the knee joint, especially after fatigue. However, whether selective fatigue of agonists or antagonist muscles would cause different changes in muscle activation patterns is unknown.HypothesisKnee extension fatigue would have a higher influence on landing biomechanics compared with a knee flexion protocol.Study designRepeated-measures design.MethodsTwenty healthy subjects (10 males and 10 females) performed two sets of repeated maximal isokinetic concentric efforts of the knee extensors (KE) at 120° s^?1 until they could no longer consistently produce 30% of maximum torque. On a separate day, a similar knee flexion (KF) fatigue protocol was also performed. Single leg landings from 30 cm drop height were performed before, in the middle and after the end of the fatigue test. The mean normalized electromyographic (EMG) signal of the vastus medialis (VM), vastus lateralis (VL), biceps femoris (BF) and gastrocnemius (GAS) at selected landing phases were determined before, during and after fatigue. Quadriceps:hamstrings (Q:H) EMG ratio as well as sagittal hip and knee angles and vertical ground reaction force (GRF) were also recorded.ResultsTwo-way analysis of variance designs showed that KE fatigue resulted in significantly lower GRF and higher knee flexion angles at initial contact while maximum hip and knee flexion also increased (p < 0.05). This was accompanied by a significant decline of BF EMG, unaltered EMG of vastii and GAS muscles and increased Q:H ratio. In contrast, KF fatigue had no effects on vGRFs but it was accompanied by increased activation of VM, BF and GAS while the Q:H increased during before landing and decreased after impact.ConclusionFatigue responses during landing are highly dependent on the muscle which is fatigued.     

The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.     

Integrated single‐cell analysis shows Pichia pastoris secretes protein stochastically

The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single‐cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell‐cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner. Biotechnol. Bioeng. 2010;106: 319–325. © 2010 Wiley Periodicals, Inc.     

Newly isolated bacterial strains belonging to Bacillaceae (Bacillus sp.) and Micrococcaceae accelerate death of the honey bee mite, Varroa destructor (V. jacobsoni), in laboratory assays

Newly isolated bacterial strains belonging to Bacillaceae (Bacillus sp.), Micrococcaceae and three unidentified strains were tested for their pathogenicity against the mite, Varroa destructor. The Bacillus sp. strain and two of the strains belonging to the Micrococcaceae family significantly decreased the time for 50% mortality of the mite population (up to 57%) and hence may be potential control agents. In in vitro bioassay whole cells, extracellular broth and cellular extract of the Bacillus sp. strain effectively killed the mites, suggesting that both endotoxins and exotoxins contributed to the killing.     

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5’ region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.     

Ectopic expression of clusterin／apolipoprotein J or Bcl-2 decreases thesensitivity of HaCaT cells to toxic effects of ropivacaine

Local anesthetics inhibit cell proliferation and induce apoptosis in various cell types. Ropivacaine, a unique, novel tertiary amine-type anesthetic, was shown to inhibit the proliferation of several cell types including keratinocytes. We found that Ropivacaine could inhibit the proliferation and induce apoptosis in an immortalized human keratinocyte line,HaCaT, in a dose- and time-dependent manner and with the deprivation of serum. The dose-dependent induction of apoptosis by ropivacaine was demonstrated by DNA fragmentation analysis and the proteolytic cleavage of a caspase-3 substrate—poly (ADP-ribose) polymerase (PARP). In addition, ropivacaine downregulated the expression of clusterin/ apoliporotein J, a protein with anti-apoptotic properties, in a dose-dependent manner, which well correlated with the induction of apoptosis of HaCaT cells. To investigate the role of clusterin/apoliporotein J in ropivacaine-induced apoptosis,HaCaT cells overexpressing clusterin/apoliporotein J were generated and compared to cells expressing the well established anti-apoptotic Bcl-2 protein. Ectopic overexpression of the secreted form of clusterin/apoliporotein J or Bcl-2decreased the sensitivity of HaCaT cells to toxic effects of ropivacaine as demonstrated by DNA fragmentation, the proteolytic cleavage of PARP and by a reduction in procaspase-3 expression. Furthermore, the downregulation of endogenous clusterin/apolipoprotein J levels by ropivacaine suggested that this might be one mechanism by which ropivacaine induced cell death in HaCaT cells. In conclusion, the ability of ropivacaine to induce antiproliferative responses and to suppress the expression of the anti-apoptotic protein clusterin/apolipoprotein J, combined with previously reported anti-inflammatory activity and analgesic property of the drug, suggests that ropivacaine may have potential utility in the local treatment of tumors.     

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1)

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.