Interleukin (IL)-6 is recognised as an important cytokine involved in inflammatory diseases of the central nervous system (CNS).
To perform a large retrospective study designed to test cerebrospinal fluid (CSF) IL-6 levels in the context of neurological diseases, and evaluate its usefulness as a biomarker to help discriminate multiple sclerosis (MS) from other inflammatory neurological diseases (OIND).
Patients and Methods
We analyzed 374 CSF samples for IL-6 using a quantitative enzyme-linked immunosorbent assay. Groups tested were composed of demyelinating diseases of the CNS (DD, n = 117), including relapsing-remitting MS (RRMS, n = 65), primary progressive MS (PPMS, n = 11), clinically isolated syndrome (CIS, n = 11), optic neuritis (ON, n = 30); idiopathic transverse myelitis (ITM, n = 10); other inflammatory neurological diseases (OIND, n = 35); and non-inflammatory neurological diseases (NIND, n = 212). Differences between groups were analysed using Kruskal−Wallis test and Mann−Whitney U-test.
CSF IL-6 levels exceeded the positivity cut-off of 10 pg/ml in 18 (51.4%) of the 35 OIND samples, but in only three (3.9%) of the 76 MS samples collected. CSF IL-6 was negative for all NIND samples tested (0/212). IL-6 cut-off of 10 pg/ml offers 96% sensitivity to exclude MS.
CSF IL-6 may help to differentiate MS from its major differential diagnosis group, OIND.
The ability of IFN-β to induce IL-10 production from innate immune cells is important for its anti-inflammatory properties and is believed to contribute to its therapeutic value in treating multiple sclerosis patients. In this study, we identified that IFN-β stimulates IL-10 production by activating the JAK1- and PI3K-signaling pathways. JAK1 activity was required for IFN-β to activate PI3K and Akt1 that resulted in repression of glycogen synthase kinase 3 (GSK3)-β activity. IFN-β–mediated suppression of GSK3-β promoted IL-10, because IL-10 production by IFN-β–stimulated dendritic cells (DC) expressing an active GSK3-β knockin was severely reduced, whereas pharmacological or genetic inhibition of GSK3-β augmented IL-10 production. IFN-β increased the phosphorylated levels of CREB and STAT3 but only CREB levels were affected by PI3K. Also, a knockdown in CREB, but not STAT3, affected the capacity of IFN-β to induce IL-10 from DC. IL-10 production by IFN-β–stimulated DC was shown to suppress IFN-γ and IL-17 production by myelin oligodendrocyte glycoprotein-specific CD4+ T cells, and this IL-10–dependent anti-inflammatory effect was enhanced by directly targeting GSK3 in DC. These findings highlight how IFN-β induces IL-10 production and the importance that IL-10 plays in its anti-inflammatory properties, as well as identify a therapeutic target that could be used to increase the IL-10–dependent anti-inflammatory properties of IFN-β.
Ability to accurately determine time of stroke onset remains challenging. We hypothesized that an early biomarker characterized by a rapid increase in blood after stroke onset may help defining better the time window during which an acute stroke patient may be candidate for intravenous thrombolysis or other intravascular procedures.
The blood level of 29 proteins was measured by immunoassays on a prospective cohort of stroke patients (N = 103) and controls (N = 132). Mann-Whitney U tests, ROC curves and diagnostic odds ratios were applied to evaluate their clinical performances.
Among the 29 molecules tested, GST-π concentration was the most significantly elevated marker in the blood of stroke patients (p<0.001). More importantly, GST-π displayed the best area under the curve (AUC, 0.79) and the best diagnostic odds ratios (10.0) for discriminating early (N = 22, <3 h of stroke onset) vs. late stroke patients (N = 81, >3 h after onset). According to goal-oriented distinct cut-offs (sensitivity(Se)-oriented: 17.7 or specificity(Sp)-oriented: 65.2 ug/L), the GST-π test obtained 91%Se/50%Sp and 50%Se/91%Sp, respectively. Moreover, GST-π showed also the highest AUC (0.83) and performances for detecting patients treated with tPA (N = 12) compared to ineligible patients (N = 103).
This study demonstrates that GST-π can accurately predict the time of stroke onset in over 50% of early stroke patients. The GST-π test could therefore complement current guidelines for tPA administration and potentially increase the number of patients accessing thrombolysis.
The influence of rumen microbial structure and functions on host physiology remains poorly understood. This study aimed to investigate the interaction between the ruminal microflora and the host by correlating bacterial diversity with fermentation measurements and feed efficiency traits, including dry matter intake, feed conversion ratio, average daily gain, and residual feed intake, using culture-independent methods. Universal bacterial partial 16S rRNA gene products were amplified from ruminal fluid collected from 58 steers raised under a low-energy diet and were subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Multivariate statistical analysis was used to relate specific PCR-DGGE bands to various feed efficiency traits and metabolites. Analysis of volatile fatty acid profiles showed that butyrate was positively correlated with daily dry matter intake (P < 0.05) and tended to have higher concentration in inefficient animals (P = 0.10), while isovalerate was associated with residual feed intake (P < 0.05). Our results suggest that particular bacteria and their metabolism in the rumen may contribute to differences in host feed efficiency under a low-energy diet. This is the first study correlating PCR-DGGE bands representing specific bacteria to metabolites in the bovine rumen and to host feed efficiency traits.
The dental plaque is comprised of numerous bacterial species which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of IL-1β, IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential.
Materials and Methods
HGECs were challenged with the four bacterial species, live or heat-killed, at various MOIs (multiplicity of infection) and the elicited IL-1β, IL-6, IL-8 and IL-10 responses were assayed by ELISA.
Primary HGECs challenged with live P. gingivalis produced high levels of IL-1β, while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory.
We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary human gingival epithelial cells that may reflect their individual virulence or commensal status.
P. gingivalis; A. actinomycetemcomitans; F. nucleatum; S.gordonii; epithelial cells; cytokines
Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human β-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon Gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood.
Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3β (GSK-3β) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3β by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3β enhanced the induction of β-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3β (S9A) abrogated HBD-2 whereas kinase inactive GSK-3β (R85A) induced higher amounts of HBD-2.
These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3β and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential.
IL-1 receptor antagonist (IL-1Ra), a natural inhibitor of IL-1β, has been shown to regulate the progression of a variety of inflammatory diseases. Although experimental studies and clinical trials have demonstrated the importance of IL-1Ra in chronic inflammatory diseases, the cellular mechanisms responsible for regulating the endogenous production of IL-1Ra by innate immune cells are currently unresolved. In the present study, we identify that glycogen-synthase kinase 3 (GSK3) regulates the production of the anti-inflammatory cytokine IL-1Ra via its ability to regulate the MAPK ERK1/2 in TLR-stimulated cells. Elucidation of the cell-signaling pathway by which GSK3 controlled ERK activity demonstrated that GSK3 inhibition resulted in an abrogation in the levels of the inhibitory residue serine 71 on Rac1 and increased the ability of Rac1 to interact with and activate p21-activated protein kinase. siRNA-mediated knockdown of Rac1 attenuated the ability of GSK3 inhibition to augment phopsho-ERK1/2 levels in LPS-stimulated immune cells. Moreover, inhibiting the ability of GSK3 to augment ERK1/2 activity abrogated enhanced IL-1Ra production by GSK3-inhibited cells. Our findings identify that GSK3 negatively regulates the levels of IL-1Ra produced by LPS-stimulated innate immune cells.
TLR 4 stimulation of innate immune cells induces a MyD88-independent signaling pathway that leads to the production of IFN-β. In this study, we demonstrate glycogen synthase kinase 3-β (GSK3-β) plays a fundamental role in this process. Suppression of GSK3-β activity by either pharmacological inhibition, small interfering RNA-mediated gene silencing, or ectopic expression of a kinase-dead GSK3-β mutant enhanced IFN-β production by TLR4-stimulated macrophages. Conversely, ectopic expression of a constitutively active GSK3-β mutant severely attenuated IFN-β production. GSK3-β was found to negatively control the cellular levels of the transcription factor c-Jun and its nuclear association with ATF-2. Small interfering RNA-mediated knockdown of c-Jun levels abrogated the ability of GSK3-β inhibition to augment IFN-β, demonstrating that the ability of GSK3 to control IFN-β production was due to its ability to regulate c-Jun levels. The ability of GSK3 inhibition to control IFN-β production was confirmed in vivo as mice treated with a GSK3 inhibitor exhibited enhanced systemic levels of IFN-β upon LPS challenge. These findings identify a novel regulatory pathway controlling IFN-β production by TLR4-stimulated innate immune cells.
Signals induced by the TCR and CD28 costimulatory pathway have been shown to lead to the inactivation of the constitutively active enzyme, glycogen synthase kinase-3 (GSK3), which has been implicated in the regulation of IL-2 and T cell proliferation. However, it is unknown whether GSK3 plays a similar role in naive and memory CD4+ T cell responses. Here we demonstrate a divergence in the dependency on the inactivation of GSK3 in the proliferative responses of human naive and memory CD4+ T cells. We find that although CD28 costimulation increases the frequency of phospho-GSK3 inactivation in TCR-stimulated naive and memory CD4+ T cells, memory cells are less reliant on GSK3 inactivation for their proliferative responses. Rather we find that GSK3β plays a previously unrecognized role in the selective regulation of the IL-10 recall response by human memory CD4+ T cells. Furthermore, GSK3β-inactivated memory CD4+ T cells acquired the capacity to suppress the bystander proliferation of CD4+ T cells in an IL-10-dependent, cell contact-independent manner. Our findings reveal a dichotomy present in the function of GSK3 in distinct human CD4+ T cell populations.
IL-12p70 is an immunoregulatory cytokine that has been shown to induce IL-10 production from CD4+ T cells, yet the underlying cellular mechanisms controlling this process are poorly understood. In the present study, we demonstrate that IL-12p70 induces IL-10 production from human memory CD4+ T cells via a PI3K-dependent signaling mechanism. Specifically, stimulation of human memory CD4+ T cells in the presence of IL-12p70 lead to increased PI3K activity and the subsequent phosphorylation and inactivation of the downstream constitutively active serine/threonine kinase, glycogen synthase kinase-3β (GSK3β). Inhibition of PI3K prevented the inactivation of GSK3β by IL-12p70, as well as the subsequent ability of IL-12p70 to augment IL-10 levels by memory CD4+ T cells. Moreover, ectopic expression of a constitutively active form of GSK3β abrogated the ability of IL-12p70 to increase IL-10 production by TCR-stimulated CD4+ T cells. In contrast, direct inhibition of GSK3 mimicked the effect of IL-12p70 on IL-10 production by memory CD4+ T cells. Analysis of downstream transcription factors identified that the ability of IL-12p70 to inactivate GSK3β lead to increased levels of c-Jun. The ability of IL-12p70 to inactivate GSK3β and induce c-Jun levels was required for IL-12 to augment IL-10 production by human memory CD4+ T cells, since small interfering RNA-mediated gene silencing of c-Jun abrogated this process. These studies identify the cellular mechanism by which IL-12 induces IL-10 production from human memory CD4+ T cells.
Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria.
We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes.
We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.
Human DNA polymerase η (Pol η) modulates susceptibility to skin cancer by promoting translesion DNA synthesis (TLS) past sunlight-induced cyclobutane pyrimidine dimers. Despite its well-established role in TLS synthesis, the role of Pol η in maintaining genome stability in the absence of external DNA damage has not been well explored. We show here that short hairpin RNA-mediated depletion of Pol η from undamaged human cells affects cell cycle progression and the rate of cell proliferation and results in increased spontaneous chromosome breaks and common fragile site expression with the activation of ATM-mediated DNA damage checkpoint signaling. These phenotypes were also observed in association with modified replication factory dynamics during S phase. In contrast to that seen in Pol η-depleted cells, none of these cellular or karyotypic defects were observed in cells depleted for Pol ι, the closest relative of Pol η. Our results identify a new role for Pol η in maintaining genomic stability during unperturbed S phase and challenge the idea that the sole functional role of Pol η in human cells is in TLS DNA damage tolerance and/or repair pathways following exogenous DNA damage.
Toll-like receptors (TLR) are pattern recognition receptors for highly conserved microbial molecular patterns. Activation of TLR is a pivotal step in the initiation of innate, inflammatory, and immune defense mechanisms. Recent findings indicate that G protein-coupled receptors (GPCR) may modulate TLR signaling, but it is unclear which GPCR are involved in this process. One such cooperation between GPCR and TLR can be attributed to the sphingosine 1-phosphate (S1P) receptor family. The S1P receptors (S1P1–5) are a family of GPCR with a high affinity for S1P, a serum-borne bioactive lipid associated with diverse biological activities such as inflammation and healing. In this study, we show that pro-inflammatory cytokine production, including IL-6 and IL-8, was increased with LPS and concomitant S1P stimulation. Furthermore, elevated cytokine production following LPS and S1P challenge in human gingival epithelial cells (HGEC) was significantly reduced when TLR4, S1P1 or S1P3 signaling was blocked. Our study also shows that S1P1 and S1P3 expression was induced by LPS in HGEC, and this elevated expression enhanced the influence of S1P in its cooperation with TLR4 to increase cytokine production. This cooperation between TLR4 and S1P1 or S1P3 demonstrates that TLR4 and GPCR can interact to enhance cytokine production in epithelial cells.
Cytokines; Epithelial cells; S1P1; S1P3; TLR4
Accurate DNA replication during S-phase is fundamental to maintain genome integrity. During this critical process, replication forks frequently encounter obstacles that impede their progression. While the regulatory pathways which act in response to exogenous replication stress are beginning to emerge, the mechanisms by which fork integrity is maintained at naturally occurring endogenous replication-impeding sequences remains obscure. Notably, little is known about how cells replicate through special chromosomal regions containing structured non-B DNA, e.g. G4 quartets, known to hamper fork progression or trigger chromosomal rearrangements. Here, we have investigated the role in this process of the human translesion synthesis (TLS) DNA polymerases of the Y-family (pol η, pol ι, and pol κ), specialized enzymes known to synthesize DNA through DNA damage. We show that depletion by RNA interference of expression of the genes for Pol η or Pol κ, but not Pol ι, sensitizes U2OS cells treated with the G4-tetraplex interactive compound telomestatin and triggers double-strand breaks in HeLa cells harbouring multiple copies of a G-rich sequence from the promoter region of the human c-MYC gene, chromosomally integrated as a transgene. Moreover, we found that downregulation of Pol κ only raises the level of DSB in HeLa cells containing either one of two breakage hotspot structured DNA sequences in the chromosome, the major break region (Mbr) of BCL-2 gene and the GA rich region from the far right-hand end of the genome of the Kaposi Sarcoma associated Herpesvirus. These data suggest that naturally occurring DNA structures are physiological substrates of both pol η and pol κ. We discuss these data in the light of their downregulation in human cancers.
DNA Replication; Genetic Instability; Double Strand Breaks; non-B DNA
The oral pathogen Porphyromonas gingivalis has been shown to modulate apoptosis in different cell types, but its effect on epithelial cells remains unclear.
We demonstrate that primary human gingival epithelial cells (HGECs) challenged with live P. gingivalis for 24 hours exhibit apoptosis, and we characterize this by M30 epitope detection, caspase-3 activity, DNA fragmentation and Annexin-V staining. Live bacteria strongly upregulated intrinsic and extrinsic apoptotic pathways. Pro-apoptotic molecules such as caspase-3, -8, -9, Bid and Bax were upregulated after 24 hours. The anti-apoptotic Bcl-2 was also upregulated, but this was not sufficient to ensure cell survival. The main P. gingivalis proteases arginine and lysine gingipains are necessary and sufficient to induce host cell apoptosis. Thus, live P. gingivalis can invoke gingival epithelial cell apoptosis in a time and dose dependent manner with significant apoptosis occurring between 12 and 24 hours of challenge via a gingipain-dependent mechanism.
The present study provides evidence that live, but not heat-killed, P. gingivalis can induce apoptosis after 24 hours of challenge in primary human gingival epithelial cells. Either arginine or lysine gingipains are necessary and sufficient factors in P. gingivalis elicited apoptosis.
Anemia is a common feature of chronic kidney disease, but the management of anemia in children is complex. Erythropoietin and supplemental iron are used to maintain hemoglobin levels. The National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (NKF-KDOQI) clinical practice guidelines for the management of anemia specifically in children were recently published. Pediatric nephrologists are encouraged to use current clinical practice guidelines and best evidence in conjunction with their clinical experience to optimally manage patients with anemia.
Chronic kidney disease; Erythropoietin; Hemoglobin; Iron; Pediatric
Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1α and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1α/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1α stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1) in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo) cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1α/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1α-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion) supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1α/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor). These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.
Relationships between click-evoked otoacoustic emissions (CEOAEs) and behavioral thresholds have not been explored above 5 kHz due to limitations in CEOAE measurement procedures. New techniques were used to measure behavioral thresholds and CEOAEs up to 16 kHz. A long cylindrical tube of 8-mm diameter, serving as a reflection-less termination, was used to calibrate audiometric stimuli and design a wideband CEOAE stimulus. A second click was presented 15 dB above a probe click level that varied over a 44 dB range, and a nonlinear residual procedure extracted a CEOAE from these click responses. In some subjects (age 14-29 years) with normal hearing up to 8 kHz, CEOAE spectral energy and latency were measured up to 16 kHz. Audiometric thresholds were measured using an adaptive yes-no procedure. Comparison of CEOAE and behavioral thresholds suggested a clinical potential of using CEOAEs to screen for high-frequency hearing loss. CEOAE latencies determined from the peak of averaged, filtered, temporal envelopes decreased to 1 ms with increasing frequency up to 16 kHz. Individual CEOAE envelopes included both compressively-growing, longer-delay components consistent with a coherent-reflection source, and linearly- or expansively-growing, shorter-delay components consistent with a distortion source. Envelope delays of both components were approximately invariant with level.
43.64.Jb; 43.66.Yw; 43.64.Kc
Stimulus frequency otoacoustic emissions (SFOAEs) measured using a suppressor tone in human ears are analogous to two-tone suppression responses measured mechanically and neurally in mammalian cochleae. SFOAE suppression was measured in 24 normal-hearing adults at octave frequencies (fp=0.5–8.0 kHz) over a 40 dB range of probe levels (Lp). Suppressor frequencies (fs) ranged from −2.0 to 0.7 octaves re: fp, and suppressor levels ranged from just detectable suppression to full suppression. The lowest suppression thresholds occurred for “best” fs slightly higher than fp. SFOAE growth of suppression (GOS) had slopes close to one at frequencies much lower than best fs, and shallow slopes near best fs, which indicated compressive growth close to 0.3 dB/dB. Suppression tuning curves constructed from GOS functions were well defined at 1, 2, and 4 kHz, but less so at 0.5 and 8.0 kHz. Tuning was sharper at lower Lp with an equivalent rectangular bandwidth similar to that reported behaviorally for simultaneous masking. The tip-to-tail difference assessed cochlear gain, increasing with decreasing Lp and increasing fp at the lowest Lp from 32 to 45 dB for fp from 1 to 4 kHz. SFOAE suppression provides a noninvasive measure of the saturating nonlinearities associated with cochlear amplification on the basilar membrane.
Periodontitis is a chronic human inflammatory disease initiated and sustained by dental plaque microorganisms. A major contributing pathogen is Porphyromonas gingivalis, a gram-negative bacterium recognized by Toll-like receptor 2 (TLR2) and TLR4, which are expressed by human gingival epithelial cells (HGECs). However, it is still unclear how these cells respond to P. gingivalis and initiate inflammatory and immune responses. We have reported previously that HGECs produce a wide range of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-α), and IL-1β. In this study, we show that IL-1β has a special role in the modulation of other inflammatory cytokines in HGECs challenged with P. gingivalis. Our results show that the increased production of IL-1β correlates with the cell surface expression of TLR4, and more specifically, TLR4-normal HGECs produce fourfold more IL-1β than do TLR4-deficient HGECs after challenge. Moreover, blocking the IL-1β receptor greatly reduces the production of “secondary” proinflammatory cytokines such as IL-8 or IL-6. Our data indicate that the induction of IL-1β plays an important role in mediating the release of other proinflammatory cytokines from primary human epithelial cells following challenge with P. gingivalis, and this process may be an inflammatory enhancement mechanism adopted by epithelial cells.
Humans develop periodontitis in response to challenge by microbial dental plaque. Inflammation begins after perturbation of gingival epithelial cells by subgingival bacteria interacting through pattern-recognition receptors, including the Toll-like receptors (TLR). Porphyromonas gingivalis is a major periodontopathogen that interacts with epithelial cells through its cell surface fimbriae (FimA), leading to colonization and/or invasion. Previous work by our group has established membrane CD14 as an essential coreceptor for TLR2-mediated activation of transfected cell lines by P. gingivalis FimA. We have shown that gingival epithelial cells express TLR2 but not CD14 on their cell surfaces. We thus speculated that P. gingivalis FimA does not readily activate epithelial innate immune responses but rather functions to promote P. gingivalis colonization in the absence of a vigorous FimA-induced response. This hypothesis was verified by the findings that primary human gingival epithelial cells responded poorly to FimA in terms of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha responses, in stark contrast to the marked response to other TLR2 agonists (Pam3Cys, FSL-1) that are not strictly dependent on CD14. On the other hand, CD14-expressing human primary monocytes responded with high levels of the same cytokines to both FimA and the control TLR2 agonists. The gingival epithelial cells failed to respond to FimA even in the presence of exogenously added soluble CD14. These data indicate that the gingival epithelial cell hyporesponsiveness to FimA is attributable to the lack of membrane-expressed but not soluble CD14. In conclusion, P. gingivalis FimA differentially activates human monocytes and epithelial cells, perhaps reflecting different tactics used by P. gingivalis when interacting with different host cell types or a host strategy to limit inflammation.
Efficacy of thrombolysis in acute ischemic stroke is strongly related to physician’s ability to make an accurate diagnosis and to intervene within 3–6 h after event onset. In this context, the discovery and validation of very early blood markers have recently become an urgent, yet unmet, goal of stroke research. Ubiquitin fusion degradation protein 1 is increased in human postmortem CSF, a model of global brain insult, suggesting that its measurement in blood may prove useful as a biomarker of stroke.
Enzyme-linked immunosorbent assay (ELISA) was used to measure UFD1 in plasma and sera in three independent cohorts, European (Swiss and Spanish) and North-American retrospective analysis encompassing a total of 123 consecutive stroke and 90 control subjects.
Highly significant increase of ubiquitin fusion degradation protein 1 (UFD1) was found in Swiss stroke patients with 71% sensitivity (95% CI, 52–85.8%), and 90% specificity (95% CI, 74.2–98%) (N = 31, p < 0.0001). Significantly elevated concentration of this marker was then validated in Spanish (N = 39, p < 0.0001, 95% sensitivity (95% CI, 82.7–99.4%)), 76% specificity (95% CI, 56.5–89.7%)) and North-American stroke patients (N = 53, 62% sensitivity (95% CI, 47.9–75.2%), 90% specificity (95% CI, 73.5–97.9%), p < 0.0001). Its concentration was increased within 3 h of stroke onset, on both the Swiss (p < 0.0001) and Spanish (p = 0.0004) cohorts.
UFD1 emerges as a reliable plasma biomarker for the early diagnosis of stroke, and in the future, might be used in conjunction with clinical assessments, neuroimaging and other blood markers.
Diagnosis; Plasma Markers; Stroke; UFD1; Brain Damage
To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome.
In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides.
Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.
The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G1/S border and throughout the S phase and was negligible in both G0 and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase α. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an ∼600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.