Cellular expression and crystal structure of the murine cytomegalovirus MHC-Iv glycoprotein, m153

Mouse cytomegalovirus (MCMV), a β-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted MHC-I structure. Functions attributed to some family members include downregulation of host MHC-I (m152) and NKG2D ligands (m145, m152, m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require β2m or peptide, and is expressed at the surface of MCMV-infected cells. Its 2.4 Å crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended amino terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family

Cellular Expression and Crystal Structure of the Murine Cytomegalovirus Major Histocompatibility Complex Class I-like Glycoprotein, m153

Mouse cytomegalovirus (MCMV), a β-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted MHC-I structure. Functions attributed to some family members include downregulation of host MHC-I (m152) and NKG2D ligands (m145, m152, m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require β2m or peptide, and is expressed at the surface of MCMV-infected cells. Its 2.4 Å crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended amino terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family

Killer-cell immunoglobulin-like receptor genotyping and HLA killer-cell immunoglobulin-like receptor-ligand identification by real-time polymerase chain reaction.

The effector function of natural killer (NK) cells is modulated by surface expression of a range of killer-cell immunoglobulin-like receptors (KIRs) that interact with human leukocyte antigen (HLA) class I ligands. We describe the use of real-time polymerase chain reaction (PCR) assays that allow easy and quick detection of 16 KIR genes and the presence/absence of KIR-ligands based on allelic discrimination at codon 80 in the HLA-A/B Bw4 and HLA-C C1/C2 genes. These methods overcome the tedious and expensive nature of conventional KIR genotyping and HLA class I typing using sequence-specific primer (SSP) PCR, sequence-specific oligonucleotide (SSO) hybridization or sequence-based typing (SBT). Using these two cost-effective assays, we measured the frequencies of KIRs, KIR-ligands and KIR/KIR-ligand pairs in a cohort of Black women recruited in South Africa

Schengen-pathway controls spatially separated and chemically distinct lignin deposition in the endodermis

Lignin is a complex polymer precisely deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS

Interactions between lymphocytes and myeloid cells regulate pro- versus anti-tumor immunity

Tumor-associated myeloid cells have been implicated in regulating many of the “hallmarks of cancer” and thus fostering solid tumor development and metastasis. However, the same innate leukocytes also participate in anti-tumor immunity and restraint of malignant disease. While many factors regulate the propensity of myeloid cells to promote or repress cancerous growths, polarized adaptive immune responses by B and T lymphocytes have been identified as regulators of many aspects of myeloid cell biology by specifically regulating their functional capabilities. Here, we detail the diversity of heterogeneous B and T lymphocyte populations and their impacts on solid tumor development through their abilities to regulate myeloid cell function in solid tumors

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Studying the intra and intermolecular processes of biomolecules using two dimensional infrared spectroscopy

This Thesis focuses on using ultrafast time-resolved infrared spectroscopy and two-dimensional infrared spectroscopy (2D-IR) for determining the structure and dynamics of a system. Chapter 1 gives an introduction to 2D-IR spectroscopy which is a technique that involves vibrationally exciting a sample, with an initial IRpump pulse, then after a waiting time the sample is probed using an IRprobe pulse. A spectrum is obtained by scanning the frequency of the IRpump pulse at a fixed waiting time. This technique allows the multiple transitions to be probed and valuable information (e.g. the coupling of vibrational modes) such as how energy is transferred, both within a molecule and to its environment, to be elucidated. Chapter 2 describes investigations of the photodissociation of Fe(CO)5. This was chosen as a model system to investigate controlling the process of photodissociation using a vibrational excitation in solution. Transient 2D-IR spectroscopy (T-2D-IR) is a powerful tool, as electronic and vibrational pump pulses can be combined together with a sensitive IR probe to interrogate such processes on the ultrafast timescale. The T-2D-IR spectra of Fe(CO)5 in heptane and CH2Cl2 are presented and we demonstrate that photolysis of Fe(CO)5 can be controlled using a vibrational excitation such that the yield of the photoproducts can be altered. We find that for Fe(CO)5 in heptane, exciting either vibrational mode 2 ps before photolysis by a 266 nm UV pulse results in a small increase in the formation of Fe(CO)3. Interestingly for CH2Cl2, exciting only the lower vibrational mode 2 ps before photolysis by a 266 nm UV pulse results in decrease in the formation Fe(CO)3, no change is observed if the higher vibrational mode is excited. No change in the formation of Fe(CO)3 was also observed when the sample was immediately excited with an IRpump pulse after the UVpump pulse. These observations are discussed and the process of vibrationally controlling the photodissociation of Fe(CO)5 is clearly a very complex problem which requires further work to understand the underlying mechanism. Chapter 3 discusses the use of 2D-IR spectroscopy to investigate the gelation mechanism of carrageenan. Polysaccharide gels are a very important component in the food, pharmaceutical and cosmetic industries. In the food industry these gels act as thickeners and stabilisers. They help give products their structure and physical properties such as texture. Carrageenan is one such polysaccharide gel and contains organosulfate groups. It has been shown that it limits ice crystals growth in frozen foods which is crucial to the stability of the product. There are three different types: ι, κ and λ-carrageenan, each of these differ in the number and position of the organosulfate functional groups. The interactions in and around these functional groups are thought to be critical to the properties of the gel and the gelation mechanism. 2D-IR spectroscopy was used to directly probe these functional groups and how their interactions change as gelation occurs. For ι-carrageenan in the solution state, the organosulfate groups exist in a large broad range of environments. Upon gelation, cross peaks were observed in the 2D-IR spectra, indicating that the organosulfate groups exist in three main conformations which all interact with each other. The different environments are thought to relate to different cation interactions. These distinct environments were not observed for κ-carrageenan. We found that the spectral slices remained similar at different temperatures and this was interpreted to be due to the structural differences between ι- carrageenan and κ-carrageenan, with the latter only having one organosulfate group per monomer. The solvent dynamics of carrageenan was also investigated using ferrocyanide as a probe molecule by measuring the spectral diffusion using 2D-IR. The energy of water H-bonds in carrageenan was determined to be approximately half that of bulk water. This was thought to be because of disrupted H-bonding networks. While the bond reformation time was estimated to be approximately four times that of bulk water and was assumed to be due to confined water bonding networks. There was no discernible difference between the two types of carrageenan. Chapter 4 investigated the interaction between salivary mucins and tea polyphenols. 2D-IR spectroscopy was used to probe the interactions of the milk protein, β -lactoglobulin, and the green tea polyphenol, epigallocatechin gallate, specifically using the amide I band in order to observe any conformational changes of the protein. The N-terminal domain of MUC5B (NT5B) has been previously shown to interact the most with theaflavins, specifically theaflavin digallate. Again, using 2D-IR spectroscopy, the secondary structure of NT5B was determined to be formed of mainly β-sheet and some α-helix. When NT5B and theaflavin digallate interacted, conformational changes were observed. A reduction in β-sheet was observed while the α-helix conformation remained largely unchanged. There was also an indication of formation of aggregates and the results were interpreted to indicate that the β -sheet conformation is potentially important in the process of mucins cross linking. Chapter 5 outlines the details of the equipment and experimental approaches used in the Thesis, and two appendices contain other work I have been associated with during my PhD. Appendix B contains the investigation of tryptophan to heme electron transfer in myoglobin, using TRIR spectroscopy where the experimental data had been previously collected with my work undertaking all the analysis and interpretation of these results. Appendix C details an investigation of lignin formation, which is a complex biopolymer, and is important in the formation and function of cell walls. Confocal Raman microscopy was used to show chemically distinct lignin was produced when altering a cell signalling pathway, where, again the experimental Raman data had been previously collected with my work undertaking the multivariate analysis and interpretation of these results

Studying the intra and intermolecular processes of biomolecules using two dimensional infrared spectroscopy

This Thesis focuses on using ultrafast time-resolved infrared spectroscopy and two-dimensional infrared spectroscopy (2D-IR) for determining the structure and dynamics of a system. Chapter 1 gives an introduction to 2D-IR spectroscopy which is a technique that involves vibrationally exciting a sample, with an initial IRpump pulse, then after a waiting time the sample is probed using an IRprobe pulse. A spectrum is obtained by scanning the frequency of the IRpump pulse at a fixed waiting time. This technique allows the multiple transitions to be probed and valuable information (e.g. the coupling of vibrational modes) such as how energy is transferred, both within a molecule and to its environment, to be elucidated. Chapter 2 describes investigations of the photodissociation of Fe(CO)5. This was chosen as a model system to investigate controlling the process of photodissociation using a vibrational excitation in solution. Transient 2D-IR spectroscopy (T-2D-IR) is a powerful tool, as electronic and vibrational pump pulses can be combined together with a sensitive IR probe to interrogate such processes on the ultrafast timescale. The T-2D-IR spectra of Fe(CO)5 in heptane and CH2Cl2 are presented and we demonstrate that photolysis of Fe(CO)5 can be controlled using a vibrational excitation such that the yield of the photoproducts can be altered. We find that for Fe(CO)5 in heptane, exciting either vibrational mode 2 ps before photolysis by a 266 nm UV pulse results in a small increase in the formation of Fe(CO)3. Interestingly for CH2Cl2, exciting only the lower vibrational mode 2 ps before photolysis by a 266 nm UV pulse results in decrease in the formation Fe(CO)3, no change is observed if the higher vibrational mode is excited. No change in the formation of Fe(CO)3 was also observed when the sample was immediately excited with an IRpump pulse after the UVpump pulse. These observations are discussed and the process of vibrationally controlling the photodissociation of Fe(CO)5 is clearly a very complex problem which requires further work to understand the underlying mechanism. Chapter 3 discusses the use of 2D-IR spectroscopy to investigate the gelation mechanism of carrageenan. Polysaccharide gels are a very important component in the food, pharmaceutical and cosmetic industries. In the food industry these gels act as thickeners and stabilisers. They help give products their structure and physical properties such as texture. Carrageenan is one such polysaccharide gel and contains organosulfate groups. It has been shown that it limits ice crystals growth in frozen foods which is crucial to the stability of the product. There are three different types: ι, κ and λ-carrageenan, each of these differ in the number and position of the organosulfate functional groups. The interactions in and around these functional groups are thought to be critical to the properties of the gel and the gelation mechanism. 2D-IR spectroscopy was used to directly probe these functional groups and how their interactions change as gelation occurs. For ι-carrageenan in the solution state, the organosulfate groups exist in a large broad range of environments. Upon gelation, cross peaks were observed in the 2D-IR spectra, indicating that the organosulfate groups exist in three main conformations which all interact with each other. The different environments are thought to relate to different cation interactions. These distinct environments were not observed for κ-carrageenan. We found that the spectral slices remained similar at different temperatures and this was interpreted to be due to the structural differences between ι- carrageenan and κ-carrageenan, with the latter only having one organosulfate group per monomer. The solvent dynamics of carrageenan was also investigated using ferrocyanide as a probe molecule by measuring the spectral diffusion using 2D-IR. The energy of water H-bonds in carrageenan was determined to be approximately half that of bulk water. This was thought to be because of disrupted H-bonding networks. While the bond reformation time was estimated to be approximately four times that of bulk water and was assumed to be due to confined water bonding networks. There was no discernible difference between the two types of carrageenan. Chapter 4 investigated the interaction between salivary mucins and tea polyphenols. 2D-IR spectroscopy was used to probe the interactions of the milk protein, β -lactoglobulin, and the green tea polyphenol, epigallocatechin gallate, specifically using the amide I band in order to observe any conformational changes of the protein. The N-terminal domain of MUC5B (NT5B) has been previously shown to interact the most with theaflavins, specifically theaflavin digallate. Again, using 2D-IR spectroscopy, the secondary structure of NT5B was determined to be formed of mainly β-sheet and some α-helix. When NT5B and theaflavin digallate interacted, conformational changes were observed. A reduction in β-sheet was observed while the α-helix conformation remained largely unchanged. There was also an indication of formation of aggregates and the results were interpreted to indicate that the β -sheet conformation is potentially important in the process of mucins cross linking. Chapter 5 outlines the details of the equipment and experimental approaches used in the Thesis, and two appendices contain other work I have been associated with during my PhD. Appendix B contains the investigation of tryptophan to heme electron transfer in myoglobin, using TRIR spectroscopy where the experimental data had been previously collected with my work undertaking all the analysis and interpretation of these results. Appendix C details an investigation of lignin formation, which is a complex biopolymer, and is important in the formation and function of cell walls. Confocal Raman microscopy was used to show chemically distinct lignin was produced when altering a cell signalling pathway, where, again the experimental Raman data had been previously collected with my work undertaking the multivariate analysis and interpretation of these results