A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.     

Prevalence and Diagnosis of Hemotrophic Mycoplasma Infection in Research Sheep and Its Effects on Hematology Variables and Erythrocyte Membrane Fragility

Hemotrophic mycoplasma (hemoplasma) infection in research sheep can confound experimental results and contribute to morbidity and mortality. Prevalence and clinicopathologic studies historically relied on blood-smear diagnosis, but systematic studies using current molecular techniques are warranted. Here we sought to report the prevalence of subclinical infection in our study population, compare diagnostic sensitivity and specificity between blood smears and a PCR assay, and determine the effects of infection on CBC variables and erythrocyte membrane fragility. We collected whole-blood samples from 111 convenience-sampled research sheep. All samples were tested for hemoplasmas by using a PCR assay, blood smears were evaluated for visual presence of hemoplasmas, and CBC and osmotic fragility assays were performed. Subclinical prevalence, according to PCR diagnosis, was 14.1% (14 of 99) in our study population. Relative to the PCR assay, blood-smear diagnosis was 8.3% sensitive and 100% specific for hemoplasma detection. Subclinical infection was associated with changes in MCV, MCHC, RBC distribution width, and absolute monocyte count. Acute infection was associated with changes in RBC mass, Hgb concentration, MCV, MCH, MCHC, and absolute lymphocyte and monocyte counts. Acute infection was associated with increased mean erythrocyte fragility compared with that in uninfected control and treated sheep. We demonstrated that hemoplasma infection is common in our study population, blood-smear evaluation is insensitive at detecting infection, and infection is associated with changes in CBC variables and increased erythrocyte membrane fragility. These findings raise concerns regarding the suitability of hemoplasma-infected sheep for biomedical research.Abbreviations: MEF, mean erythrocyte fragilityHemoplasma infection in sheep is caused by Mycoplasma ovis and ‘Candidatus M. haemovis.’^15,34 Recent work, however, suggests M. ovis and ‘Ca. Mycoplasma haemovis’ represent the same organism with 2 different copies of the 16S rRNA.¹¹ This agent, formerly called Eperythrozoon ovis, is a cell-wall–free bacterial parasite that is intimately associated with the plasma membrane of sheep erythrocytes.²⁵ It typically is considered to be nonpathogenic in chronic infection, but it occasionally is associated with hemolytic anemia during acute infection.²⁴ Hemoplasma infection can confound experimental results and contribute to morbidity and mortality in research animals.²⁰ Infections have been reported worldwide. The clinicopathologic effects in lab animals may be a concern for biomedical research.²The organism cannot be grown in culture, making its diagnosis difficult. Previous prevalence and clinicopathologic studies relied on blood-smear diagnosis or serology.^{5,6,8-10,13,14,16,20,22,25-27,31} Systematic studies on the diagnosis and clinicopathologic effects of ovine hemoplasma infection using current, molecular techniques are unavailable. In addition, the contemporary prevalence of hemoplasma infection in research sheep in the United States is unknown.The purpose of the current study was to evaluate: 1) the prevalence of subclinical hemoplasma infection in our study population of research sheep; 2) the sensitivity and specificity of blood smears to detect hemoplasma infection; 3) the effects of subclinical and acute hemoplasma infection on CBC variables; and 4) the effects of acute hemoplasma infection on erythrocyte membrane fragility. We hypothesized that: 1) subclinical hemoplasma infection is common; 2) the examination of blood smears is not as sensitive or specific as is PCR analysis for detecting hemoplasma infection; 3) subclinical and acute infection alters CBC variables; and 4) acute infection increases erythrocyte membrane fragility.To address these questions, we collected whole-blood samples from 111 convenience-sampled research sheep as part of routine health surveillance. All samples were PCR tested by using hemoplasma-specific primers, blood smears were evaluated, and CBC analyses were performed. Osmotic fragility assays were performed on a subset of animals.     

An enzyme-coupled continuous spectrophotometric assay for magnesium protoporphyrin IX methyltransferases

The second committed step in chlorophyll biosynthesis is the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to magnesium protoporphyrin IX (MgP) forming MgP monomethylester (MgPME). This reaction is catalyzed by the enzyme MgP methyltransferase (ChlM). Previous investigation of this enzyme has involved the use of time-consuming techniques requiring separation of products from substrates. More recent methyltransferase studies use coupling enzymes to monitor changes in absorption/fluorescence for the measurement of activity. However, due to the spectral properties of porphyrins, many of these assays are unsuitable for analysis of the catalytic properties of ChlM. Here we report the successful development of a coupled, continuous spectrophotometric assay to measure the activity of ChlM. The product of the methyltransferase reaction, S-adenosyl-l-homocysteine (SAH), is converted into adenine and then hypoxanthine by the recombinant coupling enzymes SAH nucleosidase and adenine deaminase, respectively. The appearance of hypoxanthine results in a decrease in absorbance at 265 nm.The utility of this assay was shown by the characterization of ChlM from the cyanobacterium Synechocystis sp. PCC 6803. Kinetic parameters obtained support data acquired using the discontinuous HPLC-based assay and provide further evidence for the stimulation of ChlM by the H subunit of magnesium chelatase (ChlH).     

Lipid droplet-organelle interactions; sharing the fats

Lipid droplets (LDs) are key cellular organelles involved in lipid storage and mobilisation. While the major signalling cascades and many of the regulators of lipolysis have been identified, the cellular interactions involved in lipid mobilisation and release remain largely undefined. In non-adipocytes, LDs are small, mobile and interact with other cellular compartments. In contrast, adipocytes primarily contain very large, immotile LDs. The striking morphological differences between LDs in adipocytes and non-adipocytes suggest that key differences must exist in the manner in which LDs in different cell types interact with other organelles. Recent studies have highlighted the complexity of LD interactions, which can be both homotypic, with each other, and heterotypic, with other organelles. The molecules involved in these interactions are also now emerging, including Rab proteins, key regulators of membrane traffic, and caveolin, an integral membrane protein providing a functional link between the cell surface and LDs. Here we summarise recent insights into the cell biology of the LD particularly focussing on the homotypic and heterotypic interactions in both adipocytes and non-adipocytes. We speculate that these interactions may involve inter-organelle membrane contact sites or a hemi-fusion type mechanism to facilitate lipid transfer.     

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets

Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH₂-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH₂-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.Chemerin is a recently discovered chemoattractant molecule that is predicted to share structural similarity with cystatins (cysteine protease inhibitors) and cathelicidin precursors (antibacterial peptides) (1). Chemerin is present in circulating blood and several human inflammatory fluids (1). Even though chemerin is not similar to CXC and CC chemokines based on primary amino acid sequence, it functions like a chemokine in that it induces leukocyte migration and intracellular calcium mobilization. Chemerin receptor chemokine-like receptor 1 (CMKLR1,³ also named ChemR23) is a G protein-coupled receptor specifically expressed by circulating human plasmacytoid dendritic cells, natural killer cells, and tissue macrophages (1–5). In their capacity as antigen-presenting cells, plasmacytoid dendritic cells and macrophages can influence the activation of many other cell types, including monocytes, myeloid dendritic cells, B cells, T cells, and natural killer cells; thus chemerin appears to be an important chemoattractant in both innate and adaptive immune responses (2, 6, 7).Chemerin circulates in blood in an inactive prochemerin form at low nanomolar concentrations (∼3 nm) (4). Its chemotactic activity is released following proteolytic cleavage of its carboxyl-terminal amino acids by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades (4, 8). These include factor XIIa, VIIa, plasmin, neutrophil elastase, and mast cell tryptase. Of interest, staphopain B, a cysteine protease secreted by Staphylococcus aureus, also cleaves prochemerin and converts it into a potent chemoattractant (9). Interestingly, the cleavage sites in the labile carboxyl terminus (NH₂-YFPGQFAFSKALPRS-COOH) are not conserved, and the cleavage products generated by chemerin-activating proteases display different potencies in bioactivity assays. Based on synthetic peptides, the 9-mer NH₂-YFPGQFAFS-COOH is the most active, but it is still not as active as intact cleaved chemerin protein, indicating that the amino-terminal part of chemerin is required for maximal activity (4, 10).Plasma carboxypeptidases CPN and CPB cleave the basic amino acids arginine or lysine from the carboxyl terminus of proteins or peptides such as bradykinin and complement proteins C3a and C5a. CPN is a constitutively active zinc metalloprotease present in plasma at a concentration of about 100 nm and is considered the major anaphylatoxins inhibitor (11), generating inactive “desArg” forms of C3a and C5a. In contrast, CPB exists in plasma as a proenzyme, proCPB, or thrombin-activable fibrinolysis inhibitor (TAFI) at a concentration of about 50 nm and is activated by thrombin in complex with thrombomodulin on the vascular endothelial surface. CPB inhibits fibrin degradation by removing carboxyl-terminal lysines from partially digested fibrin, which prevents further incorporation of fibrinolytic plasminogen and tissue plasminogen activator (12, 13). CPB is thermolabile and has a half-life of ∼15 min at 37 °C (14). We have shown that CPB also has broad substrate reactivity and is able to cleave and inactivate bradykinin, C3a, C5a, and thrombin-cleaved osteopontin (15–17). CPN and CPB may play complementary roles, with the former being constitutively active and capable of regulating systemic anaphylatoxins, and the latter activated locally at sites of vascular injury to provide site-specific anti-inflammatory control. Peptidases can also modulate the biological activity of certain chemokines (4). For example, dipeptidyl peptidase (DPP-IV/CD26), a serine protease, inactivates CXCL9, CXCL10, CXCL11, and CXCL12 by cleaving these chemokines in the amino terminus (18, 19).Platelets store a variety of potent cytokines and chemokines within α-granules that are released upon cell activation. Platelet degranulation products, particularly the leukocyte chemoattractants, which include CXCL4 (platelet factor 4), β-thromboglobulin, CCL5 (RANTES), CCL7 (monocyte chemotactic protein 3), and CXCL12 (stromal-derived factor 1), may contribute to host defense and also play a role in pathophysiologic conditions (20, 21). For example, platelet factor 4 forms complexes with heparin in blood or some glycosaminoglycans on platelet surfaces to form the major antigen implicated in heparin-induced thrombocytopenia (22, 23). Platelets not only store CXCL12 but also express its receptor CXCR4, a coreceptor for cellular entry of human immunodeficiency virus, type 1, suggesting that platelets may be involved in host defense (24).In this study, we found that plasma CPN or CPB can function in concert with plasmin to elicit and augment the chemotactic activity of prochemerin. Furthermore, we show that platelets could store and release partially active chemerin upon activation. Thus circulating CPN/CPB and platelets may contribute to regulating the bioactivity of leukocyte chemoattractant chemerin and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.     

Dicer independent small RNAs associate with telomeric heterochromatin

Small RNAs play important roles in the establishment and maintenance of heterochromatin structures. We show the presence of telomere specific small RNAs (tel-sRNAs) in mouse embryonic stem cells that are ∼24 nucleotides in length, Dicer-independent, and 2′-O-methylated at the 3′ terminus. The tel-sRNAs are asymmetric with specificity toward telomere G-rich strand, and evolutionarily conserved from protozoan to mammalian cells. Furthermore, tel-sRNAs are up-regulated in cells that carry null mutation of H3K4 methyltransferase MLL (Mll^(−/−)) and down-regulated in cells that carry null mutations of histone H3K9 methyltransferase SUV39H (Suv39h1/h2^(−/−)), suggesting that they are subject to epigenetic regulation. These results support that tel-sRNAs are heterochromatin associated pi-like small RNAs.     

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord¹ have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons². This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate^3,4 and the chemoattractive activities of Netrin1⁵ and Sonic Hedgehog (Shh)⁶ in the floor plate in the spinal cord.Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels⁷. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag1⁸. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons^3,4 as well the necessity for this signal to direct the commissural trajectory⁹.Download video file.^{(108M, mp4)}