Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach

In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time.Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E₂ (PGE₂) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i.e. 98%) contained the consensus motif [R/K][R/K/X]X[pS/pT]. When our data were compared with a large-scale Jurkat T-lymphocyte phosphoproteomics dataset containing more than 10,500 phosphosites, a minimal overlap of 0.2% was observed. This stresses the need for such targeted analyses when the interest is in a particular kinase.Our data provide a resource of likely substrates of PKA, and potentially some substrates of closely related kinases. Network analysis revealed that about half of the observed substrates have been implicated in cAMP-induced signaling. Still, the other half of the here-identified substrates have been less well characterized, representing a valuable resource for future research.The identification and quantification of protein phosphorylation under system perturbations is an integral part of systems biology (1, 2). The combination of phosphopeptide enrichment (3–6), stable isotope labeling, and high-resolution mass spectrometry (MS) methods (7–9) has become the method of choice for the identification of novel phosphorylation sites and for the quantitation of temporal dynamics within signaling networks (10, 11), allowing the behavior of thousands of phosphorylation sites to be studied in a single experiment (10, 12, 13). Nowadays, one of the most commonly adopted high-throughput phosphoproteomics strategies utilizes two consecutive separation steps: (i) an initial fractionation to reduce the sample complexity, and (ii) a phosphopeptide-specific affinity purification. Such techniques include strong cation exchange fractionation under acidic conditions (3), followed by a chelation-based method with the use of metal ions (i.e. immobilized metal ion affinity chromatography (4), metal oxide affinity chromatography (10, 14), or Ti⁴⁺ immobilized metal ion affinity chromatography (6)). Alternatives to strong cation exchange for the first sample fractionation step have also been reported, including the use of electrostatic repulsion liquid chromatography (15, 16), which is well suited for the identification of multiply phosphorylated peptides, or hydrophilic interaction chromatography (17).Although the number of detected phosphorylated peptides is nowadays impressive, these kinds of methodologies are still inclined to identify/quantify the more abundant phosphoproteins present in a sample. For example, phosphotyrosine peptides are underrepresented because of their relatively lower abundance.In order to analyze key signaling events that may occur on less abundant phosphoproteins, more targeted approaches, focused on a specific pathway or a specific post-translational modification, are thus still essential. Studies examining post-translational modifications are often based on immunoaffinity purification at the protein or peptide level using dedicated antibodies. Recent examples include the selective enrichment of acetylated lysines (18) and phosphorylated tyrosines (19, 20). More recently, the first specific methods targeting serine/threonine phosphorylation motifs using immune-affinity assays have emerged (21, 22). The advantages of targeted approaches are their potentially higher sensitivity and more specific throughput with, as a consequence, relatively faster and easier data interpretation, which make them attractive for many systems biology applications.Immunoaffinity enrichment can be applied at both the protein and the peptide level, and both have been explored to study protein tyrosine phosphorylation (23). The first one results mainly in information on total protein phosphorylation levels. The detection of the actual phosphoresidue might be hampered by the high content of unmodified peptides derived from the immune-purified phosphoprotein and its binding partners. Immunoprecipitation at the peptide level (20, 24, 25), in contrast, leads to improved phosphosite characterization, with the identification of hundreds of sites, albeit with the loss (generally) of information regarding total protein expression.To profile the dynamic regulation of phosphorylation events via mass spectrometry, stable isotope labeling is often implemented, either with the use of amino acids in cell culture (10) or via chemical peptide labeling of the proteolytic digests (26, 27). To identify low-abundant signaling events, phosphoprotein/phosphopeptide immunoprecipitation is typically performed on several milligrams of material because of the substoichiometric abundance of post-translational modifications. This may hamper the use of expensive isotope-labeling reagents such as iTRAQ or tandem mass tag reagents, given the large amount of chemicals needed. Boersema et al. (28) introduced an alternative sensitive and accurate triplex labeling approach using inexpensive reagents (i.e. formaldehyde) that is much less limited in terms of the sample type or amount. We combined this latter stable-isotope dimethyl labeling approach (27–29) with highly specific antibodies raised against a set of cAMP-dependent protein kinase (PKA) phosphorylated substrates as based on the current literature (11, 30–34). It is generally accepted that PKA phosphorylates sites with the reasonably stringent consensus motif [R/K][R/K/X]X[pS/pT]. It should be noted that this consensus motif resembles somewhat the motifs of other AGC kinases (e.g. Akt, PKG, PKC).The basicity of the PKA motifs may hamper their analysis via MS-based proteomics, especially when trypsin is used as a protease, as the peptides may become too small to be sequenced. The use of trypsin is also unfavorable in the approach presented here when attempting to immunoprecipitate peptides bearing the PKA motif. Therefore, we decided to use Lys-C in order to keep the (dominant (RRX[pS/pT])) phosphorylated motif intact. To enhance identification, we applied decision-tree MS/MS technology (9), which makes use of HCD and ETD for more efficient fragmentation, higher mass accuracy in tandem MS mode, and less background noise (35).We applied this method to screen the response of Jurkat T cells to prostaglandin E₂ (PGE₂) treatment. PGE₂ is a potent inflammatory mediator that plays an important role in several immune-regulatory actions (36). It is produced by many different cell types, including tumor cells, where carcinogenesis is associated with chronic inflammatory responses (37). PGE₂ signaling in T cells is initiated by its binding to the G protein–coupled receptors EP1, -2, -3, and -4. Signaling pathways that are initiated by PGE₂ are for the most part under control of the second messenger cyclic adenosine monophosphate (cAMP),¹ which is generated from ATP by adenylyl cyclase when PGE₂ binds to EP2 or EP4 receptors. One of the primary targets of cAMP is PKA—cAMP binding releases the catalytic subunit activating the kinase. In the current study, we efficiently enriched close to 650 phosphopeptides containing the [R/K][R/K/X]X[pS/pT] consensus motif. Almost all these sites were absent in a recently reported comprehensive phosphoproteomics dataset of Jurkat T cells (12), compiled using shotgun strong cation exchange–immobilized metal ion affinity chromatography analysis and containing ∼10,500 phosphorylation sites, illustrative of the complementarity and selectivity of our approach. The qualitative and quantitative data presented here provide a wide-ranging and credible resource of likely PKA substrates. Network analysis confirmed several established cAMP-dependent signaling nodes in our dataset, although most identified potential PKA substrates are “novel” (i.e. not previously reported and/or linked to PKA). Therefore, the dataset presented here can be considered as a comprehensive and reliable resource for future research into cAMP-related signaling.     

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses

Mannose‐capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAMobserved in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine‐induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co‐stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL‐10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low‐dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.     

Transorbital Sonographic Evaluation of Normal Optic Nerve Sheath Diameter in Healthy Volunteers in Bangladesh

Introduction

Measurement of optic nerve sheath diameter (ONSD) by ultrasound is increasingly used as a marker to detect raised intracranial pressure (ICP). ONSD varies with age and there is no clear consensus between studies for an upper limit of normal. Knowledge of normal ONSD in a healthy population is essential to interpret this measurement.

Methods

In a prospective observational study, ONSD was measured using a 15 MHz ultrasound probe in healthy volunteers in Chittagong, Bangladesh. The aims were to determine the normal range of ONSD in healthy Bangladeshi adults and children, compare measurements in males and females, horizontal and vertical beam orientations and left and right eyes in the same individual and to determine whether ONSD varies with head circumference independent of age.

Results

136 subjects were enrolled, 12.5% of whom were age 16 or under. Median ONSD was 4.41 mm with 95% of subjects in the range 4.25–4.75 mm. ONSD was bimodally distributed. There was no relationship between ONSD and age (≥4 years), gender, head circumference, and no difference in left vs right eye or horizontal vs vertical beam.

Conclusions

Ultrasonographic ONSD in Bangladeshi healthy volunteers has a narrow bimodal distribution independent of age (≥4 years), gender and head circumference. ONSD >4.75 mm in this population should be considered abnormal.     

Detection of Anorectal and Cervicovaginal Chlamydia Trachomatis Infections following Azithromycin Treatment: Prospective Cohort Study with Multiple Time-Sequential Measures of rRNA,DNA, Quantitative Load and Symptoms

Background

Determination of Chlamydia trachomatis (Ct) treatment success is hampered by current assessment methods, which involve a single post-treatment measurement only. Therefore, we evaluated Ct detection by applying multiple laboratory measures on time-sequential post-treatment samples.

Methods

A prospective cohort study was established with azithromycin-treated (1000 mg) Ct patients (44 cervicovaginal and 15 anorectal cases). Each patient provided 18 self-taken samples pre-treatment and for 8 weeks post-treatment (response: 96%; 1,016 samples). Samples were tested for 16S rRNA (TMA), bacterial load (quantitative PCR; Chlamydia plasmid DNA) and type (serovar and multilocus sequence typing). Covariates (including behavior, pre-treatment load, anatomic site, symptoms, age, and menstruation) were tested for their potential association with positivity and load at 3–8 weeks using regression analyses controlling for repeated measures.

Findings

By day 9, Ct positivity decreased to 20% and the median load to 0.3 inclusion-forming units (IFU) per ml (pre-treatment: 170 IFU/ml). Of the 35 cases who reported no sex, sex with a treated partner or safe sex with a new partner, 40% had detection, i.e. one or more positive samples from 3–8 weeks (same Ct type over time), indicating possible antimicrobial treatment failure. Cases showed intermittent positive detection and the number of positive samples was higher in anorectal cases than in cervicovaginal cases. The highest observed bacterial load between 3–8 weeks post-treatment was 313 IFU/ml, yet the majority (65%) of positive samples showed a load of ≤2 IFU/ml. Pre-treatment load was found to be associated with later load in anorectal cases.

Conclusions

A single test at 3–8 weeks post-treatment frequently misses Ct. Detection reveals intermittent low loads, with an unknown risk of later complications or transmission. These findings warrant critical re-evaluation of the clinical management of single dose azithromycin-treated Ct patients and fuel the debate on defining treatment failure. Clinicaltrials.gov Identifier: NCT01448876.     

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.     

CD98 marks a subpopulation of head and neck squamous cell carcinoma cells with stem cell properties

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98^high cells, in contrast to CD98^low cells, are able to generate tumors in immunodeficient mice, indicating that CD98^high cells have stem cell characteristics. Furthermore, the CD98^high subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98^low fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.     

Evidence That Mast Cells Are Not Required for Healing of Splinted Cutaneous Excisional Wounds in Mice

Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-Kit^W-sh/W-sh, WBB6F₁-Kit^W/W-v, and Cpa3-Cre; Mcl-1^fl/fl mice re-epithelialized splinted excisional skin wounds at rates very similar to those in the corresponding wild type or control mice. Furthermore, at the time of closure, scars were similar in the genetically mast cell-deficient mice and the corresponding wild type or control mice in both quantity of collagen deposition and maturity of collagen fibers, as evaluated by Masson’s Trichrome and Picro-Sirius red staining. These data indicate that mast cells do not play a significant non-redundant role in these features of the healing of splinted full thickness excisional cutaneous wounds in mice.     

Evaluation of Hepatitis A Vaccine in Post-Exposure Prophylaxis,The Netherlands, 2004-2012

Background

The secondary attack rate of hepatitis A virus (HAV) among contacts of cases is up to 50%. Historically, contacts were offered immunoglobulin (IG, a human derived blood product) as post-exposure prophylaxis (PEP). Amid safety concerns about IG, HAV vaccine is increasingly recommended instead. Public health authorities’ recommendations differ, particularly for healthy contacts ≥40 years old, where vaccine efficacy data is limited. We evaluated routine use of HAV vaccine as an alternative to immunoglobulin in PEP, in those considered at low risk of severe infection in the Netherlands.

Methods

Household contacts of acute HAV cases notified in Amsterdam (2004-2012) were invited ≤14 days post-exposure, for baseline anti-HAV testing and PEP according to national guidelines: immunoglobulin if at risk of severe infection, or hepatitis A vaccine if healthy and at low risk (aged <30, or, 30-50 years and vaccinated <8 days post-exposure). Incidence of laboratory confirmed secondary infection in susceptible contacts was assessed 4-8 weeks post-exposure. In a vaccinated subgroup, relative risk (RR) of secondary infection with estimated using Poisson regression.

Results

Of 547 contacts identified, 191 were susceptible to HAV. Per-protocol, 167 (87%) were vaccinated (mean:6.7 days post-exposure, standard deviation(sd)=3.3) and 24 (13%) were given immunoglobulin (mean:9.7 days post-exposure, sd=2.8). At follow-up testing, 8/112 (7%) had a laboratory confirmed infection of whom 7 were symptomatic. All secondary infections occurred in vaccinated contacts, and half were >40 years of age. In healthy contacts vaccinated per-protocol ≤8 days post-exposure, RR_{ref. ≤15 years} of secondary infection in those >40 years was 12.0 (95%CI:1.3-106.7).

Conclusions

Timely administration of HAV vaccine in PEP was feasible and the secondary attack rate was low in those <40 years. Internationally, upper age-limits for post-exposure vaccination vary. Pending larger studies, immunoglobulin should be considered PEP of choice in people >40 years of age and those vulnerable to severe disease.     

Bacteriophage Orphan DNA Methyltransferases: Insights from Their Bacterial Origin,Function, and Occurrence

Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function.