CD14 Modulates PI3K/AKT/p38-MAPK Licensing of Negative Regulators of TLR Signaling to Restrain Chronic Inflammation

Current thinking emphasizes the primacy of CD14 in facilitating TLR recognition of microbes to initiate proinflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of _Borrelia burgdorferi_ not only triggers an inflammatory response in the absence of CD14, but one that is uncontrolled as a consequence of impaired PI3K/AKT/p38-MAPK signaling and negative regulation of TLR2. CD14 deficiency results in hyperphosphorylation of AKT and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS thereby engendering a more severe and persistent inflammatory response to _B. burgdorferi_. Perturbation of this CD14/p38-MAPK-dependent mechanism of immune regulation may underlie development of infectious chronic inflammatory syndromes

Involvement of a DNA Polymerase III Subunit in the Bacterial Response to Quinolones

Quinolone treatment induces stabilized cleavage complexes (SCCs), consisting of a covalent gyrase-DNA complex, and processing of these complexes is thought to cause double-strand breaks and chromosome fragmentation. SCCs are required but not sufficient for cytotoxicity; the mechanism that converts SCCs to double-strand breaks is not clearly understood. Evidence of chromosome fragmentation due to quinolones comes from indirect measures such as sedimentation analysis of nucleoids and measurements of lysis viscosity. This work outlines a method that combines agarose plugs, conditional lysis and field inversion gel electrophoresis to allow direct visualization of chromosomal fragmentation resulting from quinolone treatment. We are able to distinguish between latent breaks within the stabilized cleavage complex and irreversible breaks that result from downstream processing.When seeking to understand the genetic requirements for quinolone-induced SOS response, we found that a dnaQ mutant has a specific defect in SOS induction following nalidixic acid. The product of dnaQ is the &epsilon; subunit of DNA polymerase III, which provides 3' &rarr; 5' exonuclease activity. In addition to the nalidixic acid-specific SOS defect, &delta;dnaQ has multiple phenotypes: slow growth, high mutation frequency, and constitutive SOS. We propose that &epsilon; has a role in the quinolone response beyond the normal proofreading function of the subunit in the polymerase III core. Using a unique transposon mutagenesis system, we created a library of dnaQ mutants with 15 base pair insertions that were scored phenotypically. We identified mutants that separated the various phenotypes, arguing strongly that &epsilon; has multiple functions. The isolation of a stable dnaQ mutant with SOS phenotypes allows the study of this function without confounding results from spurious mutations throughout the chromosome. We also isolated a novel class of SOS "hyper-inducible" mutants. Additionally, my findings with weak and strong &beta;-clamp binding mutants provides the first in vivo characterization of these &epsilon; mutants and gives insight into the SOS response following nalidixic acid treatment.</p

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

CD14 Modulates PI3K/AKT/p38-MAPK Licensing of Negative Regulators of TLR Signaling to Restrain Chronic Inflammation

AbstractCurrent thinking emphasizes the primacy of CD14 in facilitating TLR recognition of microbes to initiate proinflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is uncontrolled as a consequence of impaired PI3K/AKT/p38-MAPK signaling and negative regulation of TLR2. CD14 deficiency results in hyperphosphorylation of AKT and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS thereby engendering a more severe and persistent inflammatory response to B. burgdorferi. Perturbation of this CD14/p38-MAPK-dependent mechanism of immune regulation may underlie development of infectious chronic inflammatory syndromes.</jats:p

Inhaled Cyclosporine For The Treatment Of Bronchiolitis Obliterans Following Hematopoietic Stem Cell Transplantation (HSCT) Or Lung Transplantation

Abstract Background Bronchiolitis Obliterans Syndrome (BOS) is a late-onset non-infectious pulmonary complication of HSCT, resulting in obstructive lung disease. BOS is thought to be a manifestation of chronic graft versus host disease (cGVHD). BOS can also occur after lung transplantation, where it is believed to represent chronic rejection of the lung allograft. In both conditions, the mainstay of therapy includes augmentation of systemic immunosuppression. However, this approach has limited efficacy and is associated with deleterious consequences including an increased risk of infection and decreased graft versus tumor/leukemia effects. We investigated whether targeted, local delivery of inhaled cyclosporine could improve or stabilize lung function in BOS patients. Methods HSCT recipients with BOS, ages 10-80, were eligible if they met the following inclusion criteria: FEV1&amp;lt;75% predicted, FEV1 decline &amp;gt;10% compared to pre-transplant FEV1, no evidence of pulmonary infection as a causative etiology, and one of the following: FEV1/FVC ratio &amp;lt;70%, air-trapping seen on CT scan or RV ≥120%, or evidence of cGVHD affecting at least one other organ system. Lung transplant recipients with BOS were also eligible. Subjects were characterized as having progressive or stable disease at study entry based on their FEV1 values in the preceding 3-18 weeks. Progressive disease was defined as a ≥10% decline in FEV1, and stable disease as an increase in FEV &amp;lt;5% or a decrease &amp;lt;10%. Subjects received cyclosporine inhalation solution (CIS) 150 mg via nebulizer 3 times weekly for 6 weeks before dose escalation to 300mg three times weekly. The primary endpoint was change in FEV1 at study completion (average of week 18 and 19 values) compared to study baseline. Pharmacokinetic sampling and lung deposition studies were performed on all subjects. Lung deposition studies were performed using SPECT/CT imaging after inhalation of CIS mixed with technetium- 99m sulfur colloid. Bronchoalveolar lavage fluid and peripheral blood samples were collected at study baseline, week 9 and week 18, and were analyzed for cytokine markers and lymphocyte phenotype. Six minute walk tests (6MWT) and quality of life testing was also performed. Results Eleven subjects (nine evaluable) have been enrolled (median age: 45 years; range: 14-73). Nine subjects had HSCT-associated BOS, and 2 had lung-transplant-associated BOS. Patients were transplanted for various underlying conditions (MDS, aplastic anemia, DLBCL, MM, ALL, Gata-2 deficiency, alpha-1 anti-trypsin deficiency, and idiopathic pulmonary fibrosis). HSCT recipients received both reduced intensity (n=4) and myeloablative (n=5) conditioning, with allografts from HLA matched relatives (n=5) or unrelated donors (n=4). The median time from BOS diagnosis to study enrollment was 9 months (range: 2-37 mos). The median FEV1 at study entry was 1.11 liters (range: 0.5-2.11), with 4 subjects having progressive disease and 7 stable disease. Adverse events associated with CIS occurred in 9/10 patients and included cough, bronchospasm, and dyspnea. Most adverse events were grade 2 (range:1-3) and occurred only during the inhalation. Three subjects went off-study (patient choice) prior to completion of the 18 weeks due to adverse events and were considered non-responders. Four of the 8 (50%) HSCT- associated BOS subjects had a response, including 3 patients with progressive disease and 1 with stable disease at study entry. Among these responders, the improvements in absolute FEV1 from baseline were 19.5%, 15%, 12.7%, and 3.4% respectively (figure). Among the responding patients, 2 were also able to decrease their systemic steroid administration by 50% and 43%. An improvement in 6MWT was noted in 1/4 responders. Four responders have enrolled onto an extension protocol. The median peak systemic absorption of cyclosporine was 99 mcg/L (Range: 32-263) 20 minutes post-CIS inhalation. Lung deposition studies showed the total deposited dose averaged 12% (range: 4-20 %) of the inhaled dose. Conclusions Inhaled cyclosporine can be delivered safely and can stabilize or improve lung function in HSCT recipients with severe BOS, allowing systemic immunosuppression to be reduced. Use of local, targeted therapy with CIS resulted in minimal systemic absorption compared to typical oral administration, and achieved high drug levels in the lung tissue as demonstrated by the lung deposition results. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Inhaled Cyclosporine For The Treatment Of Bronchiolitis Obliterans Following Hematopoietic Stem Cell Transplantation (HSCT) Or Lung Transplantation

Abstract Background Bronchiolitis Obliterans Syndrome (BOS) is a late-onset non-infectious pulmonary complication of HSCT, resulting in obstructive lung disease. BOS is thought to be a manifestation of chronic graft versus host disease (cGVHD). BOS can also occur after lung transplantation, where it is believed to represent chronic rejection of the lung allograft. In both conditions, the mainstay of therapy includes augmentation of systemic immunosuppression. However, this approach has limited efficacy and is associated with deleterious consequences including an increased risk of infection and decreased graft versus tumor/leukemia effects. We investigated whether targeted, local delivery of inhaled cyclosporine could improve or stabilize lung function in BOS patients. Methods HSCT recipients with BOS, ages 10-80, were eligible if they met the following inclusion criteria: FEV110% compared to pre-transplant FEV1, no evidence of pulmonary infection as a causative etiology, and one of the following: FEV1/FVC ratio <70%, air-trapping seen on CT scan or RV ≥120%, or evidence of cGVHD affecting at least one other organ system. Lung transplant recipients with BOS were also eligible. Subjects were characterized as having progressive or stable disease at study entry based on their FEV1 values in the preceding 3-18 weeks. Progressive disease was defined as a ≥10% decline in FEV1, and stable disease as an increase in FEV <5% or a decrease <10%. Subjects received cyclosporine inhalation solution (CIS) 150 mg via nebulizer 3 times weekly for 6 weeks before dose escalation to 300mg three times weekly. The primary endpoint was change in FEV1 at study completion (average of week 18 and 19 values) compared to study baseline. Pharmacokinetic sampling and lung deposition studies were performed on all subjects. Lung deposition studies were performed using SPECT/CT imaging after inhalation of CIS mixed with technetium- 99m sulfur colloid. Bronchoalveolar lavage fluid and peripheral blood samples were collected at study baseline, week 9 and week 18, and were analyzed for cytokine markers and lymphocyte phenotype. Six minute walk tests (6MWT) and quality of life testing was also performed. Results Eleven subjects (nine evaluable) have been enrolled (median age: 45 years; range: 14-73). Nine subjects had HSCT-associated BOS, and 2 had lung-transplant-associated BOS. Patients were transplanted for various underlying conditions (MDS, aplastic anemia, DLBCL, MM, ALL, Gata-2 deficiency, alpha-1 anti-trypsin deficiency, and idiopathic pulmonary fibrosis). HSCT recipients received both reduced intensity (n=4) and myeloablative (n=5) conditioning, with allografts from HLA matched relatives (n=5) or unrelated donors (n=4). The median time from BOS diagnosis to study enrollment was 9 months (range: 2-37 mos). The median FEV1 at study entry was 1.11 liters (range: 0.5-2.11), with 4 subjects having progressive disease and 7 stable disease. Adverse events associated with CIS occurred in 9/10 patients and included cough, bronchospasm, and dyspnea. Most adverse events were grade 2 (range:1-3) and occurred only during the inhalation. Three subjects went off-study (patient choice) prior to completion of the 18 weeks due to adverse events and were considered non-responders. Four of the 8 (50%) HSCT- associated BOS subjects had a response, including 3 patients with progressive disease and 1 with stable disease at study entry. Among these responders, the improvements in absolute FEV1 from baseline were 19.5%, 15%, 12.7%, and 3.4% respectively (figure). Among the responding patients, 2 were also able to decrease their systemic steroid administration by 50% and 43%. An improvement in 6MWT was noted in 1/4 responders. Four responders have enrolled onto an extension protocol. The median peak systemic absorption of cyclosporine was 99 mcg/L (Range: 32-263) 20 minutes post-CIS inhalation. Lung deposition studies showed the total deposited dose averaged 12% (range: 4-20 %) of the inhaled dose. Conclusions Inhaled cyclosporine can be delivered safely and can stabilize or improve lung function in HSCT recipients with severe BOS, allowing systemic immunosuppression to be reduced. Use of local, targeted therapy with CIS resulted in minimal systemic absorption compared to typical oral administration, and achieved high drug levels in the lung tissue as demonstrated by the lung deposition results. Disclosures: No relevant conflicts of interest to declare

Fixation using alternative implants for the treatment of hip fractures (FAITH): design and rationale for a multi-centre randomized trial comparing sliding hip screws and cancellous screws on revision surgery rates and quality of life in the treatment of femoral neck fractures

Background Hip fractures are a common type of fragility fracture that afflict 293,000 Americans (over 5,000 per week) and 35,000 Canadians (over 670 per week) annually. Despite the large population impact the optimal fixation technique for low energy femoral neck fractures remains controversial. The primary objective of the FAITH study is to assess the impact of cancellous screw fixation versus sliding hip screws on rates of revision surgery at 24 months in individuals with femoral neck fractures. The secondary objective is to determine the impact on health-related quality of life, functional outcomes, health state utilities, fracture healing, mortality and fracture-related adverse events. Methods/Design FAITH is a multi-centre, multi-national randomized controlled trial utilizing minimization to determine patient allocation. Surgeons in North America, Europe, Australia, and Asia will recruit a total of at least 1,000 patients with low-energy femoral neck fractures. Using central randomization, patients will be allocated to receive surgical treatment with cancellous screws or a sliding hip screw. Patient outcomes will be assessed at one week (baseline), 10 weeks, 6, 12, 18, and 24 months post initial fixation. We will independently adjudicate revision surgery and complications within 24 months of the initial fixation. Outcome analysis will be performed using a Cox proportional hazards model and likelihood ratio test. Discussion This study represents major international efforts to definitively resolve the treatment of low-energy femoral neck fractures. This trial will not only change current Orthopaedic practice, but will also set a benchmark for the conduct of future Orthopaedic trials.Full Tex