Prognostic value of interleukin-1 receptor antagonist gene polymorphism and cytomegalovirus seroprevalence in patients with coronary artery disease

Background

Chronic inflammatory stimuli such as cytomegalovirus (CMV) infection and various genetic polymorphisms determining the inflammatory response are assumed to be important risk factors in atherosclerosis. We investigated whether patients with stable coronary artery disease (CAD) and homozygous for allele 2 of the interleukin 1 receptor antagonist (IL-1RA) gene and seropositive for CMV represent a group particular susceptible for recurrent cardiovascular events.

Methods

In a series of 300 consecutive patients with angiographically defined CAD a prospective follow-up was conducted (mean age 57.9 years, median follow-up time 38.2 months).

Results

No statistically significant relationship was found between CMV serostatus and IL-1RN*2 (alone or in combination) and risk for future cardiovascular events (CVE). The hazard ratio (HR) for a CVE given positive CMV-serology and IL-1RN*2 was 1.07 (95% confidence interval (CI) 0.32–3.72) in the fully adjusted model compared to seronegative CMV patients not carrying the IL-1RN*2 allele. In this prospective cohort study involving 300 patients with angiographically defined CAD at baseline, homozygousity for allele 2 of the IL-1 RA and seropositivity to CMV alone and in combination were not associated with an increased risk for cardiovascular events during follow-up; in addition, combination of the CMV-seropositivity and IL-1RN*2 allele were not associated with a proinflammatory response

Conclusion

Our study suggests that seropositivity to CMV and IL-1RA*2 genotype alone or in combination might not be a strong risk factor for recurrent cardiovascular events in patients with manifest CAD, and is not associated with levels of established inflammatory markers.     

Brain trauma enhances transient cytomegalovirus invasion of the brain only in mice that are immunodeficient

Cytomegalovirus (CMV) is one of the most common viral pathogens leading to neurological dysfunction in individuals with depressed immune systems. How CMV enters the brain remains an open question. The hypothesis that brain injury may enhance the entrance of CMV into the brain was tested. Insertion of a sterile needle into the brain caused a dramatic increase in mouse CMV in the brains of immunodeficient SCID mice inoculated peripherally within an hour of injury and examined 1 week later; peripheral inoculation 48 h after injury and a 1-week survival resulted in only a modest infection at the site of injury. In contrast, uninjured SCID mice, as well as injured immunocompetent control mice, showed little sign of viral infection at the same time intervals. Direct inoculation of the brain resulted in widespread dispersal and enhanced replication of mCMV in SCID brains tested 1 week later but not in parallel control brains. Differential viremia was unlikely to account for the greater viral load in the SCID brain, since increased mCMV in the blood of SCID compared to controls was not detected until a longer interval. These data suggest that brain injury enhances CMV invasion of the brain, but only when the adaptive immune system is compromised, and that the brain's ability to resist viral infection recovers rapidly after injury.     

Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

Background

Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.

Results

We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.

Conclusions

In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.     

Cytomegalovirus-induced embryopathology: mouse submandibular salivary gland epithelial-mesenchymal ontogeny as a model

Background  

Human studies suggest, and mouse models clearly demonstrate, that cytomegalovirus (CMV) is dysmorphic to early organ and tissue development. CMV has a particular tropism for embryonic salivary gland and other head mesenchyme. CMV has evolved to co-opt cell signaling networks so to optimize replication and survival, to the detriment of infected tissues. It has been postulated that mesenchymal infection is the critical step in disrupting organogenesis. If so, organogenesis dependent on epithelial-mesenchymal interactions would be particularly vulnerable. In this study, we chose to model the vulnerability by investigating the cell and molecular pathogenesis of CMV infected mouse embryonic submandibular salivary glands (SMGs).     

Absence of vasoactive intestinal peptide expression in hematopoietic cells enhances Th1 polarization and antiviral immunity in mice

Vasoactive intestinal peptide (VIP) induces regulatory dendritic cells (DC) in vitro that inhibit cellular immune responses. We tested the role of physiological levels of VIP on immune responses to murine CMV (mCMV) using VIP-knockout (VIP-KO) mice and radiation chimeras engrafted with syngenic VIP-KO hematopoietic cells. VIP-KO mice had less weight loss and better survival following mCMV infection compared with wild-type (WT) littermates. mCMV-infected VIP-KO mice had lower viral loads, faster clearance of virus, with increased numbers of IFN-γ(+) NK and NKT cells, and enhanced cytolytic activity of NK cells. Adaptive antiviral cellular immunity was increased in mCMV-infected VIP-KO mice compared with WT mice, with more Th1/Tc1-polarized T cells, fewer IL-10(+) T cells, and more mCMV-M45 epitope peptide MHC class I tetramer(+) CD8(+) T cells (tetramer(+) CD8 T cells). mCMV-immune VIP-KO mice had enhanced ability to clear mCMV peptide-pulsed target cells in vivo. Enhanced antiviral immunity was also seen in WT transplant recipients engrafted with VIP-KO hematopoietic cells, indicating that VIP synthesized by neuronal cells did not suppress immune responses. Following mCMV infection there was a marked upregulation of MHC-II and CD80 costimulatory molecule expression on DC from VIP-KO mice compared with DC from WT mice, whereas programmed death-1 and programmed death ligand-1 expression were upregulated in activated CD8(+) T cells and DC, respectively, in WT mice, but not in VIP-KO mice. Because the absence of VIP in immune cells increased innate and adaptive antiviral immunity by altering costimulatory and coinhibitory pathways, selective targeting of VIP signaling represents an attractive therapeutic target to enhance antiviral immunity.     

Cytomegalovirus induces interferon-stimulated gene expression and is attenuated by interferon in the developing brain

Cytomegalovirus (CMV) is considered the most common infectious agent causing permanent neurological dysfunction in the developing brain. We have previously shown that CMV infects developing brain cells more easily than it infects mature brain cells and that this preference is independent of the host B- and T-cell responses. In the present study, we examined the innate antiviral defenses against mouse (m) and human (h) CMVs in developing and mature brain and brain cells. mCMV infection induced interferon (IFN)-stimulated gene expression by 10- to 100-fold in both glia- and neuron-enriched cultures. Treatment of primary brain cultures with IFN-alpha, -beta, and -gamma or a synthetic RNA, poly(I:C), reduced the number of mCMV-infected cells, both in older cells and in fresh cultures from embryonic mouse brains. When a viral dose that killed almost all unprotected cells was used, IFN-protected cells had a natural appearance, and when they were tested with whole-cell patch clamp recording, they appeared physiologically normal with typical resting membrane potentials and action potentials. mCMV infection increased expression of representative IFN-stimulated genes (IFIT3, OAS, LMP2, TGTP, and USP18) in both neonatal and adult brains to similarly large degrees. The robust upregulation of gene expression in the neonatal brain was associated with a much higher degree of viral replication at this stage of development. In contrast to the case for downstream gene induction, CMV upregulated IFN-alpha/beta expression to a greater degree in the adult brain than in the neonatal brain. Similar to the case with cultured brain cells, IFN treatment of the developing brain in vivo depressed mCMV replication. In parallel work with cultured primary human brain cells, IFN and poly(I:C) treatment reduced hCMV infection and prevented virus-mediated cell death. These results suggest that coupling IFN administration with current treatments may reduce CMV infections in the developing brain.     

Pathogenesis and vertical transmission of a transplacental rat cytomegalovirus

Background

Cytomegalovirus (CMV) congenital infection is the major viral cause of well-documented birth defects in human. Because CMV is species-specific, the main obstacle to developing animal models for congenital infection is the difference in placental architecture, which preludes virus transmission across the placenta. The rat placenta, resembling histologically to that of human, could therefore facilitate the study of CMV congenital infection in human.

Results

In this report, we present clear evidences of the transplacental property of a new rat CMV (RCMV), namely ALL-03, which had been isolated from placenta and uterus of the house rat. Our study signifies the detection of infectious virus, virus particles, viral protein and DNA as well as immune response to demonstrate a natural model of acute CMV infection including the immunocompetent and immunocompromised host associated with or without pregnancy. It is characterized by a full range of CMV related clinical signs; lesions and anatomical virus distribution to uterus, placenta, embryo, fetus, neonate, lung, kidney, spleen, liver and salivary gland of the infected rats in addition to the virus-specific seroconversion. The preference of the virus for different organs mimics the situation in immunocompromised man. Most interestingly, the placenta was observed to be involved in the maternofetal infection and hence confirmed the hypothesis that the RCMV strain ALL-03 is capable to cross the placenta and infect the offsprings congenitally.

Conclusion

The maternal viremia leading to uterine infection which subsequently infecting to the fetus through the placenta is the most likely phenomenon of CMV vertical transmission in our study.     

Longer uncommon polyamines have a stronger defense gene-induction activity and a higher suppressing activity of Cucumber mosaic virus multiplication compared to that of spermine in Arabidopsis thaliana

Key message

Our work suggests that long chain polyamines and their derivatives are potential chemicals to control viral pathogens for crop production.

Abstract

Previously we showed that two tetraamines, spermine (Spm) and thermospermine (T-Spm), induce the expression of a subset of defense-related genes and repress proliferation of Cucumber mosaic virus (CMV) in Arabidopsis. Here we tested whether the longer uncommon polyamines (LUPAs) such as caldopentamine, caldohexamine, homocaldopentamine and homocaldohexamine have such the activity. LUPAs had higher gene induction activity than Spm and T-Spm. Interestingly the genes induced by LUPAs could be classified into two groups: the one group was most responsive to caldohexamine while the other one was most responsive to homocaldopentamine. In both the cases, the inducing activity was dose-dependent. LUPAs caused local cell death and repressed CMV multiplication more efficiently as compared to Spm. LUPAs inhibited the viral multiplication of not only avirulent CMV but also of virulent CMV in a dose-dependent manner. Furthermore, LUPAs can activate the systemic acquired resistance against CMV more efficiently as compared to Spm. When Arabidopsis leaves were incubated with LUPAs, the putative polyamine oxidase (PAO)-mediated catabolites were detected even though the conversion rate was very low. In addition, we found that LUPAs induced the expression of three NADPH oxidase genes (rbohC, rbohE and rbohH) among ten isoforms. Taken together, we propose that LUPAs activate two alternative reactive oxygen species evoked pathways, a PAO-mediated one and an NADPH-oxidase-mediated one, which lead to induce defense-related genes and restrict CMV multiplication.     

The first case of septicemia due to nontoxigenic Corynebacterium diphtheriae in Poland: case report

Background

CMV-induced vasculopathy and thrombosis have been reported, but they are rare conditions usually encountered in immunocompromised patients. However more and more complications of CMV infections are recognized in immunocompetent patients.

Case presentation

We present a case report of a previously healthy adult with cytomegalovirus infection that was complicated by tibiopopliteal deep venous thrombosis and in whom Factor V Leiden heterozygous mutation was found.

Conclusion

This new case report emphasizes the involvement of cytomegalovirus in induction of vascular thrombosis in patients with predisposing risk factors for thrombosis. It is necessary to screen for CMV infection in patients with spontaneous thrombosis and an history of fever.     

Recombinant TLR5 Agonist CBLB502 Promotes NK Cell-Mediated Anti-CMV Immunity in Mice

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.     

Endogenous jasmonic and salicylic acids levels in the Cd-hyperaccumulator Noccaea (Thlaspi) praecox exposed to fungal infection and/or mechanical stress

Key message

Sensitivity to Erysiphe in Noccaea praecox with low metal supply is related to the failure in enhancing SA. Cadmium protects against fungal-infection by direct toxicity and/or enhanced fungal-induced JA signaling.

Abstract

Metal-based defense against biotic stress is an attractive hypothesis on evolutionary advantages of plant metal hyperaccumulation. Metals may compensate for a defect in biotic stress signaling in hyperaccumulators (metal-therapy) by either or both direct toxicity to pathogens and by metal-induced alternative signaling pathways. Jasmonic acid (JA) and salicylic acid (SA) are well-established components of stress signaling pathways. However, few studies evaluate the influence of metals on endogenous concentrations of these defense-related hormones. Even less data are available for metal hyperaccumulators. To further test the metal-therapy hypothesis we analyzed endogenous SA and JA concentrations in Noccaea praecox, a cadmium (Cd) hyperaccumulator. Plants treated or not with Cd, were exposed to mechanical wounding, expected to enhance JA signaling, and/or to infection by biotrophic fungus Erysiphe cruciferarum for triggering SA. JA and SA were analyzed in leaf extracts using LC–ESI(?)–MS/MS. Plants without Cd were more susceptible to fungal attack than plants receiving Cd. Cadmium alone tended to increase leaf SA but not JA. Either or both fungal attack and mechanical wounding decreased SA levels and enhanced JA in the Cd-rich leaves of plants exposed to Cd. High leaf Cd in N. praecox seems to hamper biotic-stress-induced SA, while triggering JA signaling in response to fungal attack and wounding. To the best of our knowledge, this is the first report on the endogenous JA and SA levels in a Cd-hyperaccumulator exposed to different biotic and abiotic stresses. Our results support the view of a defect in SA stress signaling in Cd hyperaccumulating N. praecox.     

Nuclear trafficking of secreted factors and cell-surface receptors: new pathways to regulate cell proliferation and differentiation, and involvement in cancers

Background

Reversible interactions between the components of cellular signaling pathways allow for the formation and dissociation of multimolecular complexes with spatial and temporal resolution and, thus, are an important means of integrating multiple signals into a coordinated cellular response. Several mechanisms that underlie these interactions have been identified, including the recognition of specific docking sites, termed a D-domain and FXFP motif, on proteins that bind mitogen-activated protein kinases (MAPKs). We recently found that phosphatidylinositol-specific phospholipase C-γ1 (PLC-γ1) directly binds to extracellular signal-regulated kinase 2 (ERK2), a MAPK, via a D-domain-dependent mechanism. In addition, we identified D-domain sequences in several other PLC isozymes. In the present studies we sought to determine whether MAPK docking sequences could be recognized in other enzymes that metabolize phosphatidylinositols (PIs), as well as in enzymes that metabolize inositol phosphates (IPs).

Results

We found that several, but not all, of these enzymes contain identifiable D-domain sequences. Further, we found a high degree of conservation of these sequences and their location in human and mouse proteins; notable exceptions were PI 3-kinase C2-γ, PI 4-kinase type IIβ, and inositol polyphosphate 1-phosphatase.

Conclusion

The results indicate that there may be extensive crosstalk between MAPK signaling and signaling pathways that are regulated by cellular levels of PIs or IPs.     

Role of RGM coreceptors in bone morphogenetic protein signaling

Background

The repulsive guidance molecule (RGM) proteins, originally discovered for their roles in neuronal development, have been recently identified as co-receptors in the bone morphogenetic protein (BMP) signaling pathway. BMPs are members of the TGFβ superfamily of signaling cytokines, and serve to regulate many aspects of cellular growth and differentiation.

Results

Here, we investigate whether RGMa, RGMb, and RGMc play required roles in BMP and TGFβ signaling in the mouse myoblast C2C12 cell line. These cells are responsive to BMPs and are frequently used to study BMP/TGFβ signaling pathways. Using siRNA reagents to specifically knock down each RGM protein, we show that the RGM co-receptors are required for significant BMP signaling as reported by two cell-based BMP activity assays: endogenous alkaline phosphatase activity and a luciferase-based BMP reporter assay. Similar cell-based assays using a TGFβ-induced luciferase reporter show that the RGM co-receptors are not required for TGFβ signaling. The binding interaction of each RGM co-receptor to each of BMP2 and BMP12 is observed and quantified, and equilibrium dissociation constants in the low nanomolar range are reported.

Conclusion

Our results demonstrate that the RGMs play a significant role in BMP signaling and reveal that these molecules cannot functionally compensate for one another.     

Protein-based identification of quantitative trait loci associated with malignant transformation in two HER2+ cellular models of breast cancer

Background

A contemporary view of the cancer genome reveals extensive rearrangement compared to normal cells. Yet how these genetic alterations translate into specific proteomic changes that underpin acquiring the hallmarks of cancer remains unresolved. The objectives of this study were to quantify alterations in protein expression in two HER2+ cellular models of breast cancer and to infer differentially regulated signaling pathways in these models associated with the hallmarks of cancer.

Results

A proteomic workflow was used to identify proteins in two HER2 positive tumorigenic cell lines (BT474 and SKBR3) that were differentially expressed relative to a normal human mammary epithelial cell line (184A1). A total of 64 (BT474-184A1) and 69 (SKBR3-184A1) proteins were uniquely identified that were differentially expressed by at least 1.5-fold. Pathway inference tools were used to interpret these proteins in terms of functionally enriched pathways in the tumor cell lines. We observed "protein ubiquitination" and "apoptosis signaling" pathways were both enriched in the two breast cancer models while "IGF signaling" and "cell motility" pathways were enriched in BT474 and "amino acid metabolism" were enriched in the SKBR3 cell line.

Conclusion

While "protein ubiquitination" and "apoptosis signaling" pathways were common to both the cell lines, the observed patterns of protein expression suggest that the evasion of apoptosis in each tumorigenic cell line occurs via different mechanisms. Evidently, apoptosis is regulated in BT474 via down regulation of Bid and in SKBR3 via up regulation of Calpain-11 as compared to 184A1.     

Large scale analysis of pediatric antiviral CD8+ T cell populations reveals sustained, functional and mature responses

Background

Cellular immunity plays a crucial role in cytomegalovirus (CMV) infection and substantial populations of CMV-specific T cells accumulate throughout life. However, although CMV infection occurs during childhood, relatively little is know about the typical quantity and quality of T cell responses in pediatric populations.

Methods

One thousand and thirty-six people (Male/Female = 594/442, Age: 0–19 yr.; 959 subjects, 20–29 yr.; 77 subjects) were examined for HLA typing. All of 1036 subjects were tested for HLA-A2 antigen. Of 1036 subjects, 887 were also tested for HLA-A23, 24 antigens. In addition, 50 elderly people (Male/Female = 11/39, Age: 60–92 yr.) were also tested for HLA-A2 antigen. We analyzed the CD8+ T cell responses to CMV, comparing these to responses in children and young. The frequencies, phenotype and function CD8+ T cells for two imunodominant epitopes from pp65 were measured.

Results

We observed consistently high frequency and phenotypically "mature" (CD27 low, CD28 low, CD45RA+) CMV-specific CD8+ T cell responses in children, including those studied in the first year of life. These CD8+ T cells retained functionality across all age groups, and showed evidence of memory "inflation" only in later adult life.

Conclusion

CMV consistently elicits a very strong CD8+ T cell response in infants and large pools of CMV specific CD8+ T cells are maintained throughout childhood. The presence of CMV may considerably mould the CD8+ T cell compartment over time, but the relative frequencies of CMV-specific cells do not show the evidence of a population-level increase during childhood and adulthood. This contrast with the marked expansion ("inflation") of such CD8+ T cells in older adults. This study indicates that large scale analysis of peptide specific T cell responses in infants is readily possible. The robust nature of the responses observed suggests vaccine strategies aimed at priming and boosting CD8+ T cells against major pathogens (including HIV, malaria and CMV itself) could be successful in this age-group.     

Surfactant Protein-A inhibits Aspergillus fumigatus-induced allergic T-cell responses

Background

Recent studies suggest that HCV infection is associated with progressive declines in pulmonary function in patients with underlying pulmonary diseases such as asthma and chronic obstructive pulmonary disease. Few molecular studies have addressed the inflammatory aspects of HCV-associated pulmonary disease. Because IL-8 plays a fundamental role in reactive airway diseases, we examined IL-8 signaling in normal human lung fibroblasts (NHLF) in response to the HCV nucleocapsid core protein, a viral antigen shown to modulate intracellular signaling pathways involved in cell proliferation, apoptosis and inflammation.

Methods

NHLF were treated with HCV core protein and assayed for IL-8 expression, phosphorylation of the p38 MAPK pathway, and for the effect of p38 inhibition.

Results

Our studies demonstrate that soluble HCV core protein induces significant increases in both IL-8 mRNA and protein expression in a dose- and time-dependent manner. Treatment with HCV core led to phosphorylation of p38 MAPK, and expression of IL-8 was dependent upon p38 activation. Using TNFα as a co-stimulant, we observed additive increases in IL-8 expression. HCV core-mediated expression of IL-8 was inhibited by blocking gC1qR, a known receptor for soluble HCV core linked to MAPK signaling.

Conclusion

These studies suggest that HCV core protein can lead to enhanced p38- and gC1qR-dependent IL-8 expression. Such a pro-inflammatory role may contribute to the progressive deterioration in pulmonary function recently recognized in individuals chronically infected with HCV.