Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.     

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.     

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens

IntroductionWe have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA).MethodsThe autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit.ResultsTwo peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen β chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency.ConclusionsHere we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.     

Gap Dynamics in a Seagrass Landscape

We investigated gap dynamics within a shallow subtidal landscape characterized by seagrass vegetation and examined the relationship between gap formation and selected physical factors. The study was conducted over 2 y by using a biannual mapping of seagrass and water depth across an 48,800-m² area in Tampa Bay, Florida. In addition, monthly sediment deposition or erosion was recorded at 96 locations within the landscape. Gaps represented from 2.4% to 5.7% of the seagrass landscape, and all were within monospecific stands of Halodule wrightii. Gaps ranged in size from 10 to 305 m² and most frequently decreased in size over time. Most gaps were small and short lived (less than 6-mo duration), but the second age group most frequently recorded was at least 1.5 y old. No new species of seagrass invaded the gaps with Halodule replacing itself 100% of the time. Gaps were recorded over the entire range of water depths within the landscape. Neither gap area nor persistence of gaps was related to water depth. However gap area was associated positively with the number of extreme sedimentation events. Gaps originated not only from removal of interior vegetation (similar to classic gaps) but also from differential growth of the seagrass margin (similar to edaphic gaps). Distinct seasonal components to the mode of formation were detected with interior-produced gaps originating primarily in the winter and margin gaps most commonly during summer. These results combine to illustrate the importance of large-scale studies with fine-scale resolution for deciphering unique features of seagrass landscape dynamics. Our historical information suggests that a static enumeration of gaps may not provide an accurate assessment of disturbance intensity in this system, and the seagrass mosaic probably is explained best by a combination of disturbance regimes and edaphic factors, such as sediment stability. Moreover, we suggest that even in areas characterized by monospecific stands of vegetation and over short or moderate time periods, gaps indirectly may influence community structure and ecosystem function via modification of habitat arrangement. Received 17 September 1998; accepted 26 April 1999.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Polycomb group protein Bmi1 negatively regulates IL-10 expression in activated macrophages

Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

Trophodynamics of the scyphomedusae Aurelia aurita. Predation rate in relation to abundance, size and type of prey organism

Ephyra larvae and small medusae (1.7–95 mm diameter, 0.01–350mg ash-free dry wt, AFDW) of the scyphozoan jellyfish Aureliaaurita were used in predation experiments with phytoplankton(the flagellate Isochrysis galbana, 4 µm diameter, {smalltilde}6 x 10^–6 µg AFDW cell^–1), ciliates (theoligotrich Strombidium sulcatum, 28 µm diameter, {smalltilde}2 x 10^–3 µg AFDW), rotifers (Synchaeta sp.,0.5 µg AFDW individual^–1) and mixed zooplankton(mainly copepods and cladocerans, 2.1–3.1 µg AFDWindividual^–1). Phytoplankton in natural concentrations(50–200 µg C I^–1) were not utilized by largemedusae (44–95 mm diameter). Ciliates in concentrationsfrom 0.5 to 50 individuals ml^"1 were consumed by ephyra larvaeand small medusae (3–14 mm diameter) at a maximum predationrate of 171 prey day^–1, corresponding to a daily rationof 0.42%. The rotifer Synchaeta sp., offered in concentrationsof 100–600 prey I^–1, resulted in daily rations ofephyra larvae (2–5 mm diameter) between 1 and 13%. Mixedzooplankton allowed the highest daily rations, usually in therange 5–40%. Large medusae (>45 mm diameter) consumedbetween 2000 and 3500 prey organisms day^"1 in prey concentrationsexceeding 100 I^–1. Predation rate and daily ration werepositively correlated with prey abundance. Seen over a broadsize spectrum, the daily ration decreased with increased medusasize. The daily rations observed in high abundance of mixedzooplankton suggest a potential ‘scope for growth’that exceeds the growth rate observed in field populations,and this, in turn, suggests that the natural populations areusually food limited. The predicted predation rate at averageprey concentrations that are characteristic of neritic environmentscannot explain the maximum growth rates observed in field populations.It is therefore suggested that exploitation of patches of preyin high abundance is an important component in the trophodynamicsof this species. ¹Present address: University of Bergen, Department of MarineBiology, N-5065 Blomsterdalen, Norway