Nonanemic Iron Deficiency in Cardiac Surgery: A Retrospective Observational Study.

OBJECTIVES: Anemia and iron deficiency in patients having cardiac surgery increases their perioperative risk. Nonanemic iron deficiency (NAID) in this group is less well-described. We aimed to investigate the incidence and outcomes of patients with NAID undergoing cardiac surgery. DESIGN: Retrospective observational study. SETTING: A single, tertiary referral center. PARTICIPANTS: Adult patients who were preassessed and underwent cardiac surgery during the study period had data collected. We enrolled 537 patients enrolled and divided them into 4 groups according to hemoglobin and ferritin: NAID, nonanemic iron replete, iron-deficiency anemia (IDA), and non-iron-deficiency anemia. INTERVENTIONS: This study was not interventional, but assessed the impact of anemia and iron deficiency on patient outcomes. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the incidence of NAID. Secondary outcomes included the number of patients who became anemic awaiting surgery, allogeneic transfusion burden, length of stay, postoperative complications, and death. 179 of 537 patients (33.3%) had NAID. Seventeen patients (9.5%) became anemic in the NAID group compared with 7 (3.3%) in the nonanemic iron replete group while awaiting for surgery (p = 0.02). Patients with NAID were more likely to receive allogeneic transfusions (33% vs 23%; p = 0.04) and had poorer recovery of hemoglobin at follow-up (13.2 ± 1.46 g/dL vs 13.9 ± 1.46 g/dL; p < 0.001). CONCLUSIONS: NAID is common and can lead to progression to anemia and increased transfusion. Iron replacement should be considered in patients with NAID in the preoperative setting. A prospective interventional trial is required to demonstrate the benefit of being iron replete.

Inhibition of cell adhesion and immune responses in the mouse model of collagen-induced arthritis with a peptidomimetic that blocks CD2-CD58 interface interactions.

CD2 and CD58 are two important costimulatory molecules involved in generating the signal II required for normal immune signaling. However, this interaction can be targeted to be of benefit in cases of abnormal immune signaling seen in autoimmune diseases. Our objective in this study was to design a peptidomimetic (compound 7) based on a ß-strand structure of the adhesion domain of CD2 protein to inhibit CD2-CD58 protein-protein interaction and its effect on immunomodulation in the collagen-induced arthritis (CIA) model. The ability of compound 7 to bind to CD58 protein was assessed using flow cytometry. The effect of compound 7 on modulating the immune response was evaluated in an autoimmune disease using CIA in mice. The stability of compound 7 was evaluated in mouse serum using mass spectrometry. Antibody (Ab) binding inhibition studies suggested that compound 7 binds to CD58 protein. Compound 7 was successful in modulating immune responses when administered in the CIA mouse model along with reducing anti-collagen Ab levels and decreasing the level of interferon gamma (IFN-γ) relative to control treatments. Compound 7 was found to be nonimmunogenic and stable in mouse serum up to 48 h. Results suggest that compound 7 can serve as a lead compound for immunomodulation, and could be a therapeutic agent for the autoimmune disease rheumatoid arthritis (RA).

BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes.

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication.

Herpes simplex virus 1 glycoprotein M and the membrane-associated protein UL11 are required for virus-induced cell fusion and efficient virus entry.

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.

Functional hierarchy of herpes simplex virus 1 viral glycoproteins in cytoplasmic virion envelopment and egress.

Herpes simplex virus 1 (HSV-1) viral glycoproteins gD (carboxyl terminus), gE, gK, and gM, the membrane protein UL20, and membrane-associated protein UL11 play important roles in cytoplasmic virion envelopment and egress from infected cells. We showed previously that a recombinant virus carrying a deletion of the carboxyl-terminal 29 amino acids of gD (gDΔct) and the entire gE gene (ΔgE) did not exhibit substantial defects in cytoplasmic virion envelopment and egress (H. C. Lee et al., J. Virol. 83:6115-6124, 2009). The recombinant virus ΔgM2, engineered not to express gM, produced a 3- to 4-fold decrease in viral titers and a 50% reduction in average plaque sizes in comparison to the HSV-1(F) parental virus. The recombinant virus containing all three mutations, gDΔct-ΔgM2-ΔgE, replicated approximately 1 log unit less efficiently than the HSV-1(F) parental virus and produced viral plaques which were on average one-third the size of those of HSV-1(F). The recombinant virus ΔUL11-ΔgM2, engineered not to express either UL11 or gM, replicated more than 1 log unit less efficiently and produced significantly smaller plaques than UL11-null or gM-null viruses alone, in agreement with the results of Leege et al. (T. Leege et al., J. Virol. 83:896-907, 2009). Analyses of particle-to-PFU ratios, relative plaque size, and kinetics of virus growth and ultrastructural visualization of glycoprotein-deficient mutant and wild-type virions indicate that gDΔct, gE, and gM function in a cooperative but not redundant manner in infectious virion morphogenesis. Overall, comparisons of single, double, and triple mutant viruses generated in the same HSV-1(F) genetic background indicated that lack of either UL20 or gK expression caused the most severe defects in cytoplasmic envelopment, egress, and infectious virus production, followed by the double deletion of UL11 and gM.

Single dose of glycoprotein K (gK)-deleted HSV-1 live-attenuated virus protects mice against lethal vaginal challenge with HSV-1 and HSV-2 and induces lasting T cell memory immune responses.

BACKGROUND: Herpes simplex virus type-1(HSV-1) and HSV-2 are important human pathogens that cause significant ocular and urogenital complications, respectively. We have previously shown that HSV-1 virions lacking glycoprotein K (gK) are unable to enter into neurons via synaptic axonal membranes and be transported in either retrograde or anterograde manner. Here, we tested the ability of HSV-1 (F) gK-null to protect against lethal challenge with either highly virulent ocular HSV-1 (McKrae strain), or genital HSV-2 (G strain). The gK-null virus vaccine efficiently protected mice against lethal vaginal infection with either HSV-1(McKrae) or HSV-2 (G). RESULTS: Female mice were immunized via a single intramuscular injection with 106 PFU of the gK-null virus. Immunized mice were treated with Depo-Provera fourteen days after vaccination and were challenged via the vaginal route one week later. Ninety percent of mice vaccinated with the gK-null virus survived HSV-1 (McKrae) challenge, while 70% of these mice survived after HSV-2 (G) challenge. Moreover, all vaccinated mice exhibited substantially reduced disease symptoms irrespective of HSV-1 or HSV-2 challenge as compared to the mock vaccinated challenge group. T-cell memory immune responses to specific glycoprotein B (gB) and glycoprotein D (gD) peptide epitopes were detectable at 7 months post vaccination. CONCLUSIONS: These results suggest that the highly attenuated, non-neurotropic gK-null virus may be used as an effective vaccine to protect against both virulent HSV-1 and HSV-2 genital infections and induce lasting immune responses.

Oncolytic herpes simplex virus 1 encoding 15-prostaglandin dehydrogenase mitigates immune suppression and reduces ectopic primary and metastatic breast cancer in mice.

Oncolytic herpes simplex virus 1 (HSV-1) viruses armed with immunomodulatory transgenes have shown potential for enhanced antitumor therapy by overcoming tumor-based immune suppression and promoting antitumor effector cell development. Previously, we reported that the new oncolytic HSV-1 virus, OncSyn (OS), engineered to fuse tumor cells, prevented tumor growth and metastasis to distal organs in the 4T1/BALB/c immunocompetent breast cancer mouse model, suggesting the elicitation of antitumor immune responses (Israyelyan et al., Hum. Gen. Ther. 18:5, 2007, and Israyelyan et al., Virol. J. 5:68, 2008). The OSV virus was constructed by deleting the OS viral host shutoff gene (vhs; UL41) to further attenuate the virus and permit dendritic cell activation and antigen presentation. Subsequently, the OSVP virus was constructed by inserting into the OSV viral genome a murine 15-prostaglandin dehydrogenase (15-PGDH) expression cassette, designed to constitutively express 15-PGDH upon infection. 15-PGDH is a tumor suppressor protein and the primary enzyme responsible for the degradation of prostaglandin E2 (PGE2), which is known to promote tumor development. OSVP, OSV, and OS treatment of 4T1 tumors in BALB/c mice effectively reduced primary tumor growth and inhibited metastatic development of secondary tumors. OSVP was able to significantly inhibit the development and accumulation of 4T1 metastatic tumor cells in the lungs of treated mice. Ex vivo analysis of immune cells following treatment showed increased inflammatory cytokine production and the presence of mature dendritic cells for the OSVP, OSV, and OS viruses. A statistically significant decrease in splenic myeloid-derived suppressor cells (MDSC) was observed only for OSVP-treated mice. These results show that intratumoral oncolytic herpes is highly immunogenic and suggest that 15-PGDH expression by OSVP enhanced the antitumor immune response initiated by viral infection of primary tumor cells, leading to reduced development of pulmonary metastases. The availability of the OSVP genome as a bacterial artificial chromosome allows for the rapid insertion of additional immunomodulatory genes that could further assist in the induction of potent antitumor immune responses against primary and metastatic tumors.

Site-specific proteolytic cleavage of the amino terminus of herpes simplex virus glycoprotein K on virion particles inhibits virus entry.

Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). Deletion of the amino-terminal 68-amino-acid (aa) portion of gK caused a reduction in efficiency and kinetics of virus entry similar to that of the gK-null virus in comparison to the HSV-1(F) parental virus. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. Immuno-electron microscopy confirmed the presence of gK and UL20 on purified virions. Coimmunoprecipitation experiments using purified virions revealed that gK interacted with UL20, as has been shown in virus-infected cells (T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, J. Virol. 82:6310-6323, 2008). Scanning of the HSV-1(F) viral genome revealed the presence of a single putative tobacco etch virus (TEV) protease site within gD, while additional TEV predicted sites were found within the UL5 (helicase-primase helicase subunit), UL23 (thymidine kinase), UL25 (DNA packaging tegument protein), and UL52 (helicase-primase primase subunit) proteins. The recombinant virus gDΔTEV was engineered to eliminate the single predicted gD TEV protease site without appreciably affecting its replication characteristics. The mutant virus gK-V5-TEV was subsequently constructed by insertion of a gene sequence encoding a V5 epitope tag in frame with the TEV protease site immediately after gK amino acid 68. The gK-V5-TEV, R-gK-V5-TEV (revertant virus), and gDΔTEV viruses exhibited similar plaque morphologies and replication characteristics. Treatment of the gK-V5-TEV virions with TEV protease caused approximately 32 to 34% reduction of virus entry, while treatment of gDΔTEV virions caused slightly increased virus entry. These results provide direct evidence that the gK and UL20 proteins, which are genetically and functionally linked to gB-mediated virus-induced cell fusion, are structural components of virions and function in virus entry. Site-specific cleavage of viral glycoproteins on mature and fully infectious virions utilizing unique protease sites may serve as a generalizable method of uncoupling the roles of viral glycoproteins in virus entry and virion assembly.

Amino acid differences in glycoproteins B (gB), C (gC), H (gH) and L (gL) are associated with enhanced herpes simplex virus type-1 (McKrae) entry via the paired immunoglobulin-like type-2 receptor α.

BACKGROUND: Herpes simplex virus type-1 (HSV-1) enters into cells via membrane fusion of the viral envelope with plasma or endosomal membranes mediated by viral glycoproteins. HSV-1 virions attach to cell surfaces by binding of viral glycoproteins gC, gD and gB to specific cellular receptors. Here we show that the human ocular and highly neurovirulent HSV-1 strain McKrae enters substantially more efficiently into cells via the gB-specific human paired immunoglobulin-like type-2 receptor-α (hPILR-α). Comparison of the predicted amino acid sequences between HSV-1(F) and McKrae strains indicates that amino acid changes within gB, gC, gH and gL may cause increased entry via the hPILR- α receptor. RESULTS: HSV-1 (McKrae) entered substantially more efficiently than viral strain F in Chinese hamster ovary (CHO) cells expressing hPIRL-α but not within CHO-human nectin-1, -(CHO-hNectin-1), CHO-human HVEM (CHO-hHVEM) or Vero cells. The McKrae genes encoding viral glycoproteins gB, gC, gD, gH, gL, gK and the membrane protein UL20 were sequenced and their predicted amino acid (aa) sequences were compared with virulent strains F, H129, and the attenuated laboratory strain KOS. Most aa differences between McKrae and F were located at their gB amino termini known to bind with the PILRα receptor. These aa changes included a C10R change, also seen in the neurovirulent strain ANG, as well as redistribution and increase of proline residues. Comparison of gC aa sequences revealed multiple aa changes including an L132P change within the 129-247 aa region known to bind to heparan sulfate (HS) receptors. Two aa changes were located within the H1 domain of gH that binds gL. Multiple aa changes were located within the McKrae gL sequence, which were preserved in the H129 isolate, but differed for the F strain. Viral glycoproteins gD and gK and the membrane protein UL20 were conserved between McKrae and F strains. CONCLUSIONS: The results indicate that the observed entry phenotype of the McKrae strain is most likely due to a combination of increased binding to heparan sulfate receptors and enhanced virus entry via gB-mediated fusion of the viral envelope with plasma membranes.

Cytomegalovirus-infected human endothelial cells can stimulate allogeneic CD4+ memory T cells by releasing antigenic exosomes.

Human CMV infection is controlled by T cell-mediated immunity and in immunosuppressed transplant patients it is associated with acute allograft rejection as well as chronic allograft vasculopathy. CMV infects endothelial cells (EC) and it is thought that CMV-specific host immune responses to infected allograft EC contribute to rejection. In vitro, CD4(+) T cells from CMV-positive donors (but not CMV-negative donors) are readily activated by CMV-infected allogeneic EC, although it is unclear how allogeneic CMV-infected EC activate self-class II MHC-restricted memory CD4(+) T cells. In this study, we confirm that purified CD4(+) T cells from CMV(+) donors are activated by allogeneic CMV-infected EC, but find that the response is dependent upon copurified APC expressing class II MHC that are autologous to the T cells. The transfer of CMV Ags from infected EC to APC can be mediated by EC-derived exosome-like particles. These results provide a mechanism by which CMV can exacerbate allograft rejection and suggest a novel function of EC-derived exosomes that could contribute in a more general manner to immune surveillance.

Intensity and duration of intermittent exercise and recovery during a soccer match.

Soccer is a sport consisting of high-intensity intermittent exercise, with players making forays across their anaerobic threshold for tactical advantage followed by periods of recovery. The intensity and duration of these work and recovery bouts were defined during a men's soccer match using StepWatch Activity Monitors recording step rate for each 3-second period. The data were coded by custom software to separate work bouts (step rate ≥ 4) from recovery bouts (step rate < 4), and a square wave of the pattern of bouts was plotted for 5 players: center forward, central midfielder, wing midfielder, central defender, and wing defender. Four values were calculated for each work and recovery bout identified: duration, and mean, maximum, and minimum step rate (intensity). This novel technique provided detailed graphical information on the duration and exercise intensity of each position throughout the match. The center midfielder was able to sustain work and recovery bout characteristics throughout the match and appeared to recover at higher intensity levels than other players. The forward showed the consequence of accumulated fatigue late in the match and was unable to sustain the duration of high-intensity work bouts observed earlier in the match. The central defender attenuated the intensity of his work and recovery bouts late in the match staying closer to a more moderate work rate with fewer high- or low-intensity bouts. Having objective data qualifying players' work and recovery bout characteristics might prove valuable for tactical decision making, substitution timing, and for planning future training sessions.

A single intramuscular vaccination of mice with the HSV-1 VC2 virus with mutations in the glycoprotein K and the membrane protein UL20 confers full protection against lethal intravaginal challenge with virulent HSV-1 and HSV-2 strains.

Herpes Simplex Virus type-1 (HSV-1) and type-2 (HSV-2) establish life-long infections and cause significant orofacial and genital infections in humans. HSV-1 is the leading cause of infectious blindness in the western world. Currently, there are no available vaccines to protect against herpes simplex infections. Recently, we showed that a single intramuscular immunization with an HSV-1(F) mutant virus lacking expression of the viral glycoprotein K (gK), which prevents the virus from entering into distal axons of ganglionic neurons, conferred significant protection against either virulent HSV-1(McKrae) or HSV-2(G) intravaginal challenge in mice. Specifically, 90% of the mice were protected against HSV-1(McKrae) challenge, while 70% of the mice were protected against HSV-2(G) challenge. We constructed the recombinant virus VC2 that contains specific mutations in gK and the membrane protein UL20 preventing virus entry into axonal compartments of neurons, while allowing efficient replication in cell culture, unlike the gK-null virus, which has a major defect in virus replication and spread. Intramuscular injection of mice with 107 VC2 plaque forming units did not cause any significant clinical disease in mice. A single intramuscular immunization with the VC2 virus protected 100% of mice against lethal intravaginal challenge with either HSV-1(McKrae) or HSV-2(G) viruses. Importantly, vaccination with VC2 produced robust cross protective humoral and cellular immunity that fully protected vaccinated mice against lethal disease. Quantitative PCR did not detect any viral DNA in ganglionic tissues of vaccinated mice, while unvaccinated mice contained high levels of viral DNA. The VC2 virus may serve as an efficient vaccine against both HSV-1 and HSV-2 infections, as well as a safe vector for the production of vaccines against other viral and bacterial pathogens.

SIAMESE cooperates with the CDH1-like protein CCS52A1 to establish endoreplication in Arabidopsis thaliana trichomes.

Endoreplication, also known as endoreduplication, is a phyogenetically widespread modified version of the cell cycle in which DNA replication is not followed by cell division. The SIAMESE (SIM) gene of Arabidopsis thaliana encodes the founding member of a novel class of plant-specific cyclin-dependent kinase (CDK) inhibitors and is a key regulator of endoreplication during the development of trichomes (shoot epidermal hairs). Here, we have identified mutations in the CCS52A1 gene as genetic modifiers of the multicellular trichome phenotype of sim mutants. Loss-of-function ccs52A1 mutations dramatically enhance the multicellularity of sim mutants trichomes in double mutants, whereas overexpression of CCS52A1 completely suppresses the sim mutant phenotype. CCS52A1 encodes a CDH1/FZR-like protein, a class of proteins that function as activators of the anaphase-promoting complex. Unicellular ccs52A1 trichomes become multicellular upon overexpression of B-type cyclin, consistent with repression of the accumulation of mitotic cyclins in the developing trichome by CCS52A1. As these M-phase-specific cyclins are known to accumulate in sim mutant trichomes, our data suggest that CCS52A1 and SIM cooperate in repressing accumulation of mitotic cyclins to establish the trichome endocycle. Comparison with endoreplication pathways in Drosophila and mammals indicates that while these organisms all use similar components to initiate endoreplication, the components are deployed differently in each organism.

SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana.

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.

Evidence that acetyl phosphate functions as a global signal during biofilm development.

We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüss and Wolfe, 1994, Mol Microbiol 12: 973-984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild-type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co-ordinate the expression of surface structures and cellular processes involved in the initial stages of wild-type biofilm development.