Nonimmune Serum Research Articles

Role of CXCR1 (CKR-1) in Inflammation of Experimental Mesangioproliferative Glomerulonephritis

CXCR1 (CKR-1), a receptor of IL-8, is expressed in various cells including neutrophils and monocytes, both of which play a major role in proliferating glomerular diseases. We investigated time-dependent expression of CXCR1 and the effect of single-dose cyclosporine A (CsA) treatment on this expression in experimental mesangioproliferative glomerulonephritis induced by anti-thymocyte serum (ATS). Wistar rats were divided into three groups. Group 1 (control, n = 24) received non-immune serum. Group 2 (nephritis, n = 24) received ATS. Group 3 (nephritis + CsA, n = 24) received ATS and CsA concomitantly. Kidneys from six rats in each group were removed at sixth hour, 3 days, 5 days, and 7 days. ATS induced proteinuria compared to controls (p < 0.001) and CsA precluded the development of proteinuria. Glomerular inflammation and mesangial proliferation were significantly higher in ATS group than control and CsA-treated rats (p < 0.001). ATS injection caused marked interstitial inflammation that was precluded by CsA (p < 0.001). CXCR1 was not expressed in control kidneys. However, ATS induced expression of CXCR1 in both glomeruli and tubulointerstitium. CsA treatment precluded CXCR1 expression in both glomeruli and tubulointerstitium only in the first 6 h. CXCR1 may contribute to inflammation in experimental mesangioproliferative glomerulonephritis. CsA may be beneficial by inhibiting CXCR1 expression and corresponding inflammation.

PEGylation of Vesicular Stomatitis Virus Extends Virus Persistence in Blood Circulation of Passively Immunized Mice

We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of multiple myeloma, an incurable malignancy of antibody-secreting plasma cells that are specifically localized in the bone marrow. One of the presumed advantages for using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically lacking in cancer patients, which is very important for clinical success. However, our studies show that nonimmune human and mouse serum can neutralize clinical-grade VSV, reducing the titer by up to 4 log units in 60 min. In addition, we show that neutralizing anti-VSV antibodies negate the antitumor efficacy of VSV, a concern for repeat VSV administration. We have investigated the potential use of covalent modification of VSV with polyethylene glycol (PEG) or a function-spacer-lipid (FSL)-PEG construct to inhibit serum neutralization and to limit hepatosplenic sequestration of systemically delivered VSV. We report that in mice passively immunized with neutralizing anti-VSV antibodies, PEGylation of VSV improved the persistence of VSV in the blood circulation, maintaining a more than 1-log-unit increase in VSV genome copies for up to 1 h compared to the genome copy numbers for the non-PEGylated virus, which was mostly cleared within 10 min after intravenous injection. We are currently investigating if this increase in PEGylated VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse models of multiple myeloma.

Per os feeding with antisera specific to peritrophic matrix proteins stimulates larval growth in Mamestra configurata Walker (Lepidoptera: Noctuidae)

Peritrophic matrix (PM) is an acellular, porous sheath composed of chitin and proteins that lines the midgut epithelial cells. The PM serves as a barrier against food particles, pathogens and toxins. Several studies in dipteran systems revealed that antibodies specific to PM proteins retard insect development by binding to their target antigens in the PM and blocking PM pores. To test whether antisera specific to PM proteins also inhibit larval growth in a lepidopteran system, antisera specific to chitin deacetylase 1, insect intestinal mucin 2, insect intestinal mucin 4 and PM protein 1 were fed to 2nd instar Mamestra configurata Walker (Lepidoptera: Noctuidae) larvae, a major pest of Brassica plants in North America. Interestingly, all larvae feeding on antisera gained more weight than the larvae feeding on the diet containing non-immune sera or no antiserum. The anti-McPM1 and anti-McIIM4 antisera treatments showed the highest larval weight gains, followed by the anti-McCDA1 and anti-McIIM2 antisera treatments. The interaction of treatment with time was found significant by the 6th day and the interaction of concentration with time was found significant only by the 12th day. No difference was found between the larval weights from all treatments at concentrations of 1 or 4%.

Subetta treatment increases adiponectin secretion by mature human adipocytes in vitro.

Purpose. To investigate the mechanism of action in peripheral tissues of novel complex drug containing release-active dilutions of antibodies to the beta subunit of the insulin receptor and antibodies to endothelial nitric oxide synthase (Subetta), which has shown efficacy in animal models of diabetes. Methods. Human mature adipocytes were incubated either with Subetta, with one of negative controls (placebo or vehicle), with one of nonspecific controls (release-active dilutions of antibodies to cannabinoid receptor type I or release-active dilutions of rabbit nonimmune serum), or with dimethyl sulfoxide (DMSO) at 37°C in a humidified incubator at 5% CO2 for three days. Rosiglitazone was used as reference drug. Secretion of adiponectin was measured by quantitative enzyme-linked immunosorbent assay (ELISA). Results. Only Subetta significantly stimulates adiponectin production by mature human adipocytes. Nonspecific controls did not significantly affect adiponectin secretion, resulting in adiponectin levels comparable to background values of the negative controls and DMSO. Conclusion. Increasing adiponectin production in absence of insulin by Subetta probably via modulating effect on the beta subunit of the insulin receptor might serve as one of the mechanisms of the antidiabetic effect of this drug. These in vitro results give first insight on possible mechanism of action of Subetta and serve as a background for further studies.

MicroRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

Effect of passive immunization against leptin on ovarian follicular development in prepubertal mice

Leptin has been demonstrated to be essential for reproduction. However, all the relevant studies reported to date have investigated either the effect of a complete absence of leptin both centrally and peripherally, or excess leptin administration. The aim of this present study was to investigate the effect of reducing peripheral leptin concentrations on ovarian follicular development in prepubertal animals via administration of an anti-leptin antibody. Pre-pubertal female mice were administered anti-leptin antibody under the skin behind the head for four days, with or without gonadotropins, and ovaries were weighed and collected for follicle counting. Control animals were treated with non-immune serum. Passive immunization against leptin, with or without gonadotropins, resulted in a significant increase in ovarian weight compared with control ovaries. Furthermore, the ovaries from the anti-leptin group had significantly greater numbers of primary follicles per ovarian section than the control group, thus suggesting an increase in the transition of primordial to primary follicles after treatment. Interestingly, animals treated with anti-leptin plus gonadotropins had a significantly higher number of Graafian follicles in their ovaries compared with the other groups. Collectively, the results of the present study indicate that reduction of leptin in the circulation promotes ovarian follicle development in female mice, suggesting that peripheral leptin acts as an inhibitor of ovarian follicle development.

Natural IgM Mediates Complement-Dependent Uptake of Francisella tularensis by Human Neutrophils via Complement Receptors 1 and 3 in Nonimmune Serum

Abstract A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F. tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.

The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

Chaperon-like Activation of Serum-Inducible Tryptophanyl-tRNA Synthetase Phosphorylation through Refolding as a Tool for Analysis of Clinical Samples

Tryptophanyl-tRNA synthetase (TrpRS) expression alters in colorectal (CRC), pancreatic (PC), and cervical (CC) cancers. Here, phosphorylation of unfolded TrpRS and its fragments is stimulated by human cancer sera (CS; n = 13) and serum of rabbit tumor induced by Rous sarcoma virus, unaffected by donor sera (NS; 11/15) and abolished by alkaline phosphatase. At 20 years of follow-up, serum-inducible TrpRS phosphorylation found years before healthy donors (3/15) diagnosed with PC, CRC, or leukemia. I have examined a specificity of serum-inducible TrpRS phosphorylation and found, surprisingly, that serine phosphorylation of unfolded TrpRS is stimulated by anti-TrpRS rabbit antisera but is unaffected by rabbit nonimmune sera and antisera to other antigens. Anti-TrpRS immunoglobulin G (IgG) inhibits phosphorylation of full-length TrpRS and stimulates phosphorylation of its 20-kDa fragment. Phosphorylation of this fragment is stimulated also by CS but not NS. 2-Mercaptoethanol and cyclic AMP exerted synergistic inhibitory effect on TrpRS phosphorylation. Anti-TrpRS sera and casein act as chaperones increasing TrpRS phosphorylation through refolding. Histone-specific protein kinase activity in CS (n = 44) and anti-TrpRS sera was lower than that in NS (n = 11), rabbit nonimmune sera and antisera to other antigens. TrpRS inhibitors, tryptamine, and tryptophanol stimulate in vivo accumulation of enzymatically inactive, nonphosphorylated, aggregated and anti-TrpRS IgG refoldable TrpRS. Phosphorylation of postsurgical tissues (n = 18) reveals TrpRS in ovarian cancer (OVC) and CC but not in normal placenta and liver. In OVC, TrpRS phosphorylation increase correlates with elevated tryptophan-dependent ATP-inorganic pyrophosphate exchange. Although not inducing cancer, TrpRS triggers signaling concomitant with cancer.

Digestive vacuoles of Plasmodium falciparum are selectively phagocytosed by and impair killing function of polymorphonuclear leukocytes

AbstractSequestration of parasitized erythrocytes and dysregulation of the coagulation and complement system are hallmarks of severe Plasmodium falciparum malaria. A link between these events emerged through the discovery that the parasite digestive vacuole (DV), which is released together with infective merozoites into the bloodstream, dually activates the intrinsic clotting and alternative complement pathway. Complement attack occurs exclusively on the membrane of the DVs, and the question followed whether DVs might be marked for uptake by polymorphonuclear granulocytes (PMNs). We report that DVs are indeed rapidly phagocytosed by PMNs after schizont rupture in active human serum. Uptake of malaria pigment requires an intact DV membrane and does not occur when the pigment is extracted from the organelle. Merozoites are not opsonized and escape phagocytosis in nonimmune serum. Antimalarial Abs mediate some uptake of the parasites, but to an extent that is not sufficient to markedly reduce reinvasion rates. Phagocytosis of DVs induces a vigorous respiratory burst that drives the cells into a state of functional exhaustion, blunting the production of reactive oxygen species (ROS) and microbicidal activity upon challenge with bacterial pathogens. Systemic overloading of PMNs with DVs may contribute to the enhanced susceptibility of patients with severe malaria toward invasive bacterial infections.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

HLA Antibody Specification Using Single-Antigen Beads—A Technical Solution for the Prozone Effect

Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect. Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone. The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q. Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.

Variable sensitivity to complement-mediated lysis among Trypanosoma rangeli reference strains

Six reference strains of Trypanosoma rangeli from different days of growth in axenic cultures were assayed for susceptibility to complement-mediated lysis by non-immune guinea-pig serum. Their authenticity was also confirmed by isoenzyme analyses. Parasites were incubated with 25% active or 68°C-inactivated serum (37°C, 30 min) for all tests; thereafter the lysis rates were determined. Highly variable lysis percentages were observed among T. rangeli strains and in the same stock at different growing days. In a few assays, three strains (Macias, R-1625 and Choachi) presented total or very high resistance. The others (H-14, San Agustín and SC-58) were generally most susceptible, and could reach lysis rates as high as Trypanosoma cruzi. After incubation with active sera, the epimastigotes were usually the predominant stages, being followed by spheromastigotes and/or transitional forms. Those stages and trypomastigotes could also be partially susceptible. In four strains, the short epimastigotes were more resistant to lysis than the long ones. Experiments with C3-deficient serum displayed total or partial participation of the alternative-complement pathway in T. rangeli lysis. This study confirmed the variable complement sensitivity of T. rangeli, which can be related to its intraspecific heterogeneity, to the remarkable complexity of its life-cycle stages, and to the methodology employed.

Correlation of protection against Japanese encephalitis virus and JE vaccine (IXIARO ®) induced neutralizing antibody titers

Immune sera from volunteers vaccinated in a blinded Phase 3 clinical trial with JE-VAX ® and a new Japanese encephalitis virus (JEV) vaccine (IC51 or IXIARO), were tested for the ability to protect mice against lethal JEV challenge. Sera from IXIARO vaccinated subjects were pooled into four batches based on neutralizing antibody measured by plaque reduction neutralization test (PRNT 50 titer): high (∼200), medium (∼40–50), low (∼20) and negative (<10). Pooled sera from JE-VAX ® vaccinated subjects (PRNT 50 titer ∼ 55) and pooled JEV antibody negative pre-vaccination sera were used as controls. Groups of ten 6- to 7-week-old female ICR mice were injected intraperitoneally with 0.5 ml of each serum pool diluted 1:2 or 1:10, challenged approximately 18 h later with a lethal dose of either JEV strain SA14 (genotype III) or strain KE-093 (genotype I) and observed for 21 days. All mice in the non-immune serum groups developed clinical signs consistent with JEV infection or died, whereas high titer sera from both IXIARO and JE-VAX ® sera protected 90–100% of the animals. Statistical tests showed similar protection against both JEV strains SA14 and KE-093 and protection correlated with the anti-JEV antibody titer of IXIARO sera as measured by PRNT 50. Ex vivo neutralizing antibody titers showed that almost all mice with a titer of 10 or greater were fully protected. In a separate study, analysis of geometric mean titers (GMTs) of the groups of mice vaccinated with different doses of IXIARO and challenged with JEV SA14 provided additional evidence that titers ≥ 10 were protective.

West Nile Virus Infection, Assam, India

To the Editor: West Nile virus (WNV) is a mosquito-borne flavivirus. Sporadic infections with this virus have been found in Africa, Europe, Asia, and the United States. In humans, most infections with WNV cause subclinical or a mild influenza-like illness; encephalitis occurs in some (1). In India, antibodies against WNV were first detected in humans in Bombay in 1952 (2). Virus activity has been reported in southern, central, and western India. WNV has been isolated in India from Culex vishnui mosquitoes in Andhra Pradesh and Tamil Nadu, from Cx. quinquefasciatus mosquitoes in Maharashtra, and from humans in Karnataka State (3). Assam (26°–27°30′N, 89°58′–95°41′E) is the most populated state in northeastern India; it contains ≈50% of the 38.8 million inhabitants of northeastern India. Japanese encephalitis virus (JEV) has caused sporadic epidemics in Assam since 1976. Studies conducted during 2000–2002 in Assam showed that 187 (53.7%) of 348 persons with acute encephalitis syndrome were infected with JEV (4). JEV-negative persons also showed symptoms of neurotropic viral infection. Suspecting the presence of some other closely related flavivirus in this region, we screened samples from persons with acute encephalitis syndrome for WNV in 2006. To our knowledge, no study has been conducted on the prevalence of WNV in this region. We report WNV activity in the state of Assam in northeastern India. Ethical approval for this study was obtained from the institutional ethical committee, Regional Medical Research Center, Dibrugarh, India. A JEV vaccination campaign (SA14-14-2 vaccine) was started in Assam during May 2006. During its first phase, children 1–15 years of age in Dibrugarh and Sivasagar Districts were vaccinated. Mosquito surveillance in the study area and in an earlier study (5) identified Cx. vishnui mosquitoes. During the study period, 103 serum samples and 88 cerebrospinal fluid samples were obtained from 167 patients with acute encephalitis syndrome admitted to the Assam Medical College and Hospital in Dibrugarh, which administers to the health needs of >7 districts of Upper Assam and neighboring states of Arunachal Pradesh and Nagaland. Among the 167 patients, 124 (74.2%) were children <15 years of age. Among the 103 serum samples, 80 were positive for immunoglobulin (Ig) M against JEV (IgM monoclonal antibody–capture ELISA; National Institute of Virology, Pune, India) and 12 (11.6%) were positive for IgM against WNV (IgM antigen-capture ELISA; Panbio, Sinnamon Park, Queensland, Australia). These samples were from persons in 4 districts in Assam (Dibrugarh, Golaghat, Sivasagar, and Tinsukia) and negative for IgM against JEV (Table). Follow-up was conducted for 9 patients; 3 died, and 1 was lost to follow-up. Table Incidence of JEV and WNV infections among patients with acute encephalitis syndrome, Assam, India* Virus-neutralizing antibody titers against JEV and WNV were estimated in pig kidney epithelial cells by using JEV (isolate 733913) and WNV (isolate 68856) and a cytopathic-effect assay in 96-well tissue culture plates (6). Mouse polyclonal antibodies against JEV and WNV and nonimmune serum samples were included in the assay. Of 9 paired serum samples, 6 showed neutralizing antibody for WNV, of which 4 showed a 4-fold increase in antibody titer. The remaining 3 paired samples showed cross-reactivity with WNV (titer <80) and JEV (titer <40). All 12 WNV-infected patients had high fever and headache. Convulsions (6 patients), altered sensorium (7 patients), vomiting (5 patients), and neck rigidity (2 patients) were also observed. Signs and symptoms at the time of hospitalization and at follow-up for 6 months (at 3-month intervals) were similar for persons infected with JEV and those infected with WNV. Neurologic sequelae observed at <6 months follow-up were impaired memory (6 patients), irritable behavior (5 patients), impaired hearing (3 patients), incoherent speech and disorientation (1 patient), breathing difficulty (1 patient), impaired speech (1 patient), and quadriparesis (1 patient). We identified WNV in regions of Assam to which JEV is endemic. The finding indicates that WNV might be the cause of a substantial number of acute encephalitis syndrome cases in this region. Fever and headache were the most common signs and symptoms, as reported (7). There were 3 deaths (all children) in 13 patients. Our results corroborate a similar observation in the Kolar District of Karnataka (8). In contrast, in western countries, the attack rate and case-fatality rate for WNV infection are higher among immunocompromised elderly patients (9). Our findings may be caused by strain variations and host susceptibility to the virus. Identification of circulating genotypes of WNV and its vectors and epidemiologic studies are needed to obtain additional information on WNV infection in this region and identify WNV as a cause of acute encephalitis syndrome.

Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.

Interactions between Bordetella pertussis and the complement inhibitor factor H

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19–20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

체지방 감소 다클론 후보 항체가 비거세돈의 체중, 분 소화율 및 혈액 대사물질에 미치는 영향

Twelve pigs were used to investigate the effects of polyclonal antibody candidate against abdominal (AAb) and subcutaneous adipocyte membrane proteins (SAb) on body weight, fecaldigestibility and blood metabolites. When AAb and SAb developed by Choi et al. (2010) were injected to pigs, the numerical increase in BW (body weight) occurred at 4 weeks post-treatment, but BW for an entire period was also increased, indicating that the BW increase may not be affected by the antibodies injection. Antibodies treatment did not affect (P>0.05) fecal digestibility of dry matter, crude protein, crude fat and crude fiber. Fecal digestibility of crude ash for control (no treatment) at 2 weeks decreased, and that for non-immunized serum treatmentgroup at 4 weeks post-treatment increased, respectively (P<0.05). However, fecal digestibility of crude ash for AAb and SAb groups did not significantly change. At 4 weeks after the antibodies treatment, blood urea N concentration for AAb and SAb groups was significantly increased (P<0.05). However, these increases may not be caused by the antibodies treatment because similar pattern in blood urea N concentration occurred before the antibodies treatment. Antibodies treatment did not affect concentration of plasma glucose and triglycerides (P<0.05). Compared with control, concentration of plasma total cholesterol for AAb and SAb groups at 4 weeks post-treatment was significantly (P<0.05) decreased. This may suggest that body fat reduction possibly occurs. In conclusion, the AAb and the SAb developed by Choi et al. (2010) may have safety in nutritional physiological metabolism in pigs. Further study on in vivo fat reduction of the antibodies against abdominal and subcutaneous adipocytes of pigs should be required for fat-reduced pork production.