Stable Lateral Meniscal Posterior Root Tears Left In Situ at Time of Anterior Cruciate Ligament Reconstruction Are of Minimal Long-Term Clinical Detriment.

PURPOSE: The purpose of this study was to compare long-term patient-reported outcomes in patients undergoing anterior cruciate ligament (ACL) reconstruction with untreated stable lateral meniscal posterior root (LMPR) tears to those with an intact meniscus. METHODS: Four hundred ninety-two subjects were followed for a minimum of 15-years post-ACL reconstruction and evaluated by an International Knee Documentation Committee questionnaire. The integrity of the meniscus was classified at surgery. Patients were grouped as either untreated injury to LMPR "with stable tear" (WST) group (n = 52) or intact lateral meniscus "no tear" (NT) group (n = 440). WST group included tears where those with a root avulsion within 9 mm of insertion and parrot beak tears with the integrity of the root attachment maintained. Outcomes were compared between groups. RESULTS: ACL graft rupture occurred in 10% in the WST group and in 11% in the NT group (P = .78). For participants with an intact graft (n = 440), the mean International Knee Documentation Committee scores were 82, in the WST group, and 87, in the NT group (P = .03), with a small effect size of .32. The WST group had a worse mean pain severity score (P = .04) and higher frequency of pain (P = .03) than the NT group, but the effect size was small (P < 0.3). There was no difference in the overall knee function (P = .209) or International Knee Documentation Committee activity level (P = .882). CONCLUSION: There was no adverse clinical outcome to leaving a stable LMPR tear in situ at the time of ACL reconstruction. LMPR tears left in situ were of minimal clinically significant long-term detriment, with outcomes similar to having an intact meniscus. There is an innate desire to fix the broken, but posterior meniscal root avulsions and stable parrot beak tears within 9 mm of insertion may not require intervention. At 15 years postinjury, most patients with a stable tear left in situ continue to enjoy an active lifestyle with a pain-free knee. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling.

The kinases MSK1 and MSK2 are activated 'downstream' of the p38 and Erk1/2 mitogen-activated protein kinases. Here we found that MSK1 and MSK2 were needed to limit the production of proinflammatory cytokines in response to stimulation of primary macrophages with lipopolysaccharide. By inducing transcription of the mitogen-activated protein kinase phosphatase DUSP1 and the anti-inflammatory cytokine interleukin 10, MSK1 and MSK2 exerted many negative feedback mechanisms. Deficiency in MSK1 and MSK2 prevented the binding of phosphorylated transcription factors CREB and ATF1 to the promoters of the genes encoding interleukin 10 and DUSP1. Mice doubly deficient in MSK1 and MSK2 were hypersensitive to lipopolysaccharide-induced endotoxic shock and showed prolonged inflammation in a model of toxic contact eczema induced by phorbol 12-myristate 13-acetate. Our results establish MSK1 and MSK2 as key components of negative feedback mechanisms needed to limit Toll-like receptor-driven inflammation.

Repeat Anterior Cruciate Ligament Injury and Return to Sport in Australian Soccer Players After Anterior Cruciate Ligament Reconstruction With Hamstring Tendon Autograft.

BACKGROUND: Soccer is the most commonly played team sport in the world and a high-risk sport for anterior cruciate ligament (ACL) injury and subsequent ACL reconstruction (ACLR). PURPOSE: To assess the rate of further ACL injury in patients who have undergone ACLR with hamstring tendon autograft after soccer injuries in Australia and to determine factors associated with repeat ACL injury and return to soccer. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: From a prospectively collected database, a series of 1000 consecutive ACLRs using hamstring autografts performed in soccer players were identified. Patients were surveyed at a minimum 5 years after reconstruction, including details of further ACL injuries to either knee, return to soccer or other sports, and psychological readiness per the Anterior Cruciate Ligament Return to Sport after Injury (ACL-RSI) scale. RESULTS: Of the 862 participants reviewed, ACL graft rupture occurred in 85 (10%) and contralateral ACL rupture in 68 (8%) within 5 years after the reconstruction. The 5-year ACL graft survivorship was 94% for females and 88% for males. The survivorship of the contralateral ACL was 92% for males and 90% for females. When compared with those aged >25 years, the odds of ACL graft rupture was increased by 4 to 5 times in those aged 19 to 25 years and 3 to 7 times in those ≤18 years. Further ACL injury to the graft or contralateral knee occurred in 44% of males aged ≤18 years. Risk factors for further ACL injury were younger age at time of surgery, male sex, and return to soccer. Graft diameter did not influence ACL graft rupture rates, and 70% of patients returned to soccer after ACLR. The mean ACL-RSI score was 59, and patients who reported more fear of reinjury on this scale were less likely to have returned to soccer. CONCLUSION: The prevalence of ACL graft rupture (10%) and contralateral ACL rupture (8%) was near equivalent over 5 years in this large cohort of mostly recreational Australian soccer players. ACLR with hamstring autograft is a reliable procedure, allowing 70% of patients to return to soccer in this high-risk population. Risk factors for further ACL injury are progressively younger age at time of surgery, male sex, and return to soccer. Graft diameter was not a factor in ACL graft rupture, indicating that other factors, particularly age, are of primary importance.

The NO-responsive hemoglobins of Campylobacter jejuni: concerted responses of two globins to NO and evidence in vitro for globin regulation by the transcription factor NssR.

Campylobacter jejuni possesses NO-responsive and -detoxifying mechanisms to survive NO during transmission and pathogenesis. C. jejuni possesses two hemoglobins. The first (Cgb) is a single-domain (non-flavo)hemoglobin encoded by gene Cj1586 (cgb), mutation of which leads to hypersensitivity to S-nitrosoglutathione and NO. Transcription of cgb is induced by nitrosative stress and confers resistance to NO, presumably via a Cgb-catalyzed dioxygenase or denitrosylase reaction that converts NO and oxygen to nitrate. Expression of Cgb in response to NO is mediated via the positively-acting transcription factor NssR, which regulates expression of a small regulon that includes cgb and ctb (Cj0465c), the latter encoding the truncated hemoglobin, Ctb. The role of Ctb is unclear: it is not directly involved in NO detoxification but is implicated in oxygen delivery or metabolism. Here, we describe attempts to define a function for Ctb by examining the effects of a ctb mutation on the NO transcriptome and cgb gene expression during normoxia and hypoxia. Mutation of ctb does not elicit major compensatory transcriptomic changes but relatively minor changes in genes involved in intermediary metabolism, solute transport and signal transduction. We present and test the hypothesis that, by binding NO or O(2), Ctb dampens the response to NO under hypoxic conditions and limits cgb expression, perhaps because Cgb function (i.e. NO detoxification) requires O(2)-dependent chemistry. We report the purification of NssR and specific binding to the ctb promoter. GSNO does not affect the high affinity of NssR for the ctb promoter.

Regulation of the miR-212/132 locus by MSK1 and CREB in response to neurotrophins.

Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.

Allograft Donor Characteristics Significantly Influence Graft Rupture After Anterior Cruciate Ligament Reconstruction in a Young Active Population.

BACKGROUND: Graft selection in anterior cruciate ligament (ACL) surgery can be difficult in a young active population given their high rates of reinjury. Allografts allow for control over graft size and reduce morbidity of autograft harvest. There are mixed results about the use of allograft in the literature; however, the influence of the properties of the allograft on outcomes has not been considered. HYPOTHESIS: ACL reconstruction with allografts from older donors will have a higher rate of graft rupture when compared with allograft from young donors. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Patients (N = 211) aged 13 to 25 years underwent primary ACL reconstruction with fresh-frozen nonirradiated allograft. Four graft types were used: patellar tendon, Achilles tendon, tibialis anterior, and tibialis posterior. Details were collected on allograft donor age and sex. At a minimum of 24 months, patients were evaluated for any further injuries and subjective analysis by International Knee Documentation Committee (IKDC) questionnaire. RESULTS: ACL graft rupture occurred in 23.5%. When grafts were separated into single strand (patellar and Achilles tendon) and multistrand (tibialis anterior and posterior), there was a significantly higher rate of reinjury in the single-strand grafts (29.9% vs 11%; P = .014). Grafts from female donors aged ≥50 years had significantly higher rates of ACL graft rupture (52.6%; P = .003) with increased odds by 6.7 times when compared with grafts from male donors aged <50 years. There was no significant difference in mean IKDC scores among the groups based on the age and sex of the allograft donor. CONCLUSION: The age and sex of the allograft donor and the morphology of the graft significantly influenced the rate of ACL graft rupture in young active patients. Tendons from female donors aged ≥50 years should be avoided given the higher rerupture rates as compared with male donors of any age and younger females.

Microbial responses to nitric oxide and nitrosative stress: growth, "omic," and physiological methods.

The study of bacterial responses to nitric oxide (NO), nitrosating agents, and other agents of nitrosative stress has a short history but has rapidly produced important insights into the interactions of these agents with model microbial systems as well as pathogenic species. Several methodological problems arise in attempting to define the global responses to these agents, whether in simply measuring growth or performing "omic" experiments in which the objective is to determine the genome-wide (transcriptomic) or proteome-wide responses. The first problem is the relatively long timescale over which the experiments are conducted--minutes, hours, or days in the case of slow-growing cultures. The second problem is not unique to NO and its congeners but concerns the difficulties encountered when sensitive and comprehensive analytical techniques (such as transcriptomics) are applied to cultures whose growth and physiology are perturbed by an inhibitor. In essence, the problem is "seeing the wood for the trees." This chapter reviews briefly the state of knowledge of NO responses and mechanisms in bacteria, particularly Escherichia coli and Campylobacter jejuni. Continuous culture has several advantages for investigating the consequences of NO exposure, and this approach is outlined with examples of recent results and conclusions. The major advantage of the chemostat is establishment of a reproducible quasi-steady state in growth, in which the growth rate can be controlled and maintained. Contrary to common belief, neither the concept nor the apparatus is difficult. Commercially available and homemade systems are described with practical advice. Establishing continuous cultures paves the way for other "omic" approaches, particularly proteomics and metabolomics, which are not covered here, as their application to the field of NO biology is in its infancy. A key to the literature describing methods suitable for assessing toxicity to microbes of NO and reactive nitrogen species is given.

Beta Interferon Production Is Regulated by p38 Mitogen-Activated Protein Kinase in Macrophages via both MSK1/2- and Tristetraprolin-Dependent Pathways.

Autocrine or paracrine signaling by beta interferon (IFN-ß) is essential for many of the responses of macrophages to pathogen-associated molecular patterns. This feedback loop contributes to pathological responses to infectious agents and is therefore tightly regulated. We demonstrate here that macrophage expression of IFN-ß is negatively regulated by mitogen- and stress-activated kinases 1 and 2 (MSK1/2). Lipopolysaccharide (LPS)-induced expression of IFN-ß was elevated in both MSK1/2 knockout mice and macrophages. Although MSK1 and -2 promote the expression of the anti-inflammatory cytokine interleukin 10, it did not strongly contribute to the ability of MSKs to regulate IFN-ß expression. Instead, MSK1 and -2 inhibit IFN-ß expression via the induction of dual-specificity phosphatase 1 (DUSP1), which dephosphorylates and inactivates the mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK). Prolonged LPS-induced activation of p38 and JNK, phosphorylation of downstream transcription factors, and overexpression of IFN-ß mRNA and protein were similar in MSK1/2 and DUSP1 knockout macrophages. Two distinct mechanisms were implicated in the overexpression of IFN-ß: first, JNK-mediated activation of c-jun, which binds to the IFN-ß promoter, and second, p38-mediated inactivation of the mRNA-destabilizing factor tristetraprolin, which we show is able to target the IFN-ß mRNA.

Do globins in microaerophilic Campylobacter jejuni confer nitrosative stress tolerance under oxygen limitation?

The microaerophilic pathogen Campylobacter jejuni possesses inducible systems for resisting NO. Two globins--Cgb (a single-domain globin) and Ctb (a truncated globin)--are up-regulated in response to NO via the positively acting transcription factor NssR. Our aims were to determine whether these oxygen-binding globins also function in severely oxygen-limited environments, as in the host. At growth-limiting oxygen transfer rates, bacteria were more S-nitrosoglutathione (GSNO) sensitive, irrespective of the presence of Cgb, Ctb, or NssR. Pregrowth of cells with GSNO enhanced GSNO resistance, even in nssR and cgb mutants, but transcriptomic profiling of oxygen-limited, NO-exposed cells failed to reveal the NssR regulon. Nevertheless, globin expression in an Escherichia coli mutant lacking the NO-detoxifying flavohemoglobin Hmp showed that Cgb and Ctb consume NO aerobically or anoxically and offer some protection to respiratory inhibition by NO. The constitutively expressed nitrite reductase NrfA does not provide resistance under oxygen-limited conditions. We, therefore, hypothesize that, although Cgb and NrfA can detoxify NO, even anoxically, they are neither up-regulated nor functional under physiologically relevant oxygen-limited conditions and, second, responses to NO do not stem from trancriptional regulation.

Cross talk between the Akt and p38α pathways in macrophages downstream of Toll-like receptor signaling.

The stimulation of Toll-like receptors (TLRs) on macrophages by pathogen-associated molecular patterns (PAMPs) results in the activation of intracellular signaling pathways that are required for initiating a host immune response. Both phosphatidylinositol 3-kinase (PI3K)-Akt and p38 mitogen-activated protein kinase (MAPK) signaling pathways are activated rapidly in response to TLR activation and are required to coordinate effective host responses to pathogen invasion. In this study, we analyzed the role of the p38-dependent kinases MK2/3 in the activation of Akt and show that lipopolysaccharide (LPS)-induced phosphorylation of Akt on Thr308 and Ser473 requires p38α and MK2/3. In cells treated with p38 inhibitors or an MK2/3 inhibitor, phosphorylation of Akt on Ser473 and Thr308 is reduced and Akt activity is inhibited. Furthermore, BMDMs deficient in MK2/3 display greatly reduced phosphorylation of Ser473 and Thr308 following TLR stimulation. However, MK2/3 do not directly phosphorylate Akt in macrophages but act upstream of PDK1 and mTORC2 to regulate Akt phosphorylation. Akt is recruited to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the membrane, where it is activated by PDK1 and mTORC2. Analysis of lipid levels in MK2/3-deficient bone marrow-derived macrophages (BMDMs) revealed a role for MK2/3 in regulating Akt activity by affecting availability of PIP3 at the membrane. These data describe a novel role for p38α-MK2/3 in regulating TLR-induced Akt activation in macrophages.

Regulation of miRNA transcription in macrophages in response to Candida albicans.

Macrophages detect pathogens via pattern recognition receptors (PRRs), which trigger several intracellular signaling cascades including the MAPK and NFκB pathways. These in turn mediate the up-regulation of pro-inflammatory cytokines that are essential to combat the pathogen. However as the over-production of pro-inflammatory cytokines results in tissue damage or septic shock, precise control of these signaling pathways is essential and achieved via the induction of multiple negative feedback mechanisms. miRNAs are small regulatory RNAs that are able to affect protein expression, via the regulation of either mRNA stability or translation. Up-regulation of specific miRNAs could have the potential to modulate PRR signaling, as has been shown for both miR-146 and miR-155. Here we have analysed which miRNAs are up-regulated in mouse macrophages in response to the fungal pathogen heat killed Candida albicans and compared the profile to that obtained with the TLR4 ligand LPS. We found that in addition to miR-146 and miR-155, both Candida albicans and LPS were also able to up-regulate miR-455 and miR-125a. Analysis of the signaling pathways required showed that NFκB was necessary for the transcription of all 4 pri-miRNAs, while the ERK1/2 and p38 MAPK pathways were also required for pri-miR-125a transcription. In addition the anti-inflammatory cytokine IL-10 was found to be able to induce miR-146a and b, but inhibited miR-155 induction. These results suggest that miR-455, miR-125, miR-146 and miR-155 may play important roles in regulating macrophage function following PRR stimulation.

Carbon monoxide-releasing antibacterial molecules target respiration and global transcriptional regulators.

Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO)(3)Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation.

Oxygen- and NssR-dependent globin expression and enhanced iron acquisition in the response of campylobacter to nitrosative stress.

Pathogenic bacteria experience nitrosative stress from NO generated in the host and from nitrosating species such as S-nitrosoglutathione. The food-borne pathogen Campylobacter jejuni responds by activating gene expression from a small regulon under the control of the NO-sensitive regulator, NssR. Here, we describe the full extent of the S-nitrosoglutathione response using transcriptomic and proteomic analysis of batch- and chemostat-cultured C. jejuni. In addition to the NssR regulon, which includes two hemoglobins (Cgb and Ctb), we identify more than 90 other up-regulated genes, notably those encoding heat shock proteins and proteins involved in oxidative stress tolerance and iron metabolism/transport. Up-regulation of a subset of these genes, including cgb, is also elicited by NO-releasing compounds. Mutation of the iron-responsive regulator Fur results in insensitivity of growth to NO, suggesting that derepression of iron-regulated genes and augmentation of iron acquisition is a physiological response to nitrosative damage. We describe the effect of oxygen availability on nitrosative stress tolerance; cells cultured at higher rates of oxygen diffusion have elevated levels of hemoglobins, are more resistant to inhibition by NO of both growth and respiration, and consume NO more rapidly. The oxygen response is mediated by NssR. Thus, in addition to NO detoxification catalyzed by the hemoglobins Cgb and possibly Ctb, C. jejuni mounts an extensive stress response. We suggest that inhibition of respiration by NO may increase availability of oxygen for Cgb synthesis and function.