Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.Lipopolysaccharides (LPS) are among the most potent inflammatory bacterial mediators and have been strongly implicated in the inflammatory response associated with gram-negative sepsis. Most LPS signaling studies have used LPS preparations derived from species within the Enterobacteriaceae, which possess relatively well-conserved lipid A structures (reviewed in reference 36). A convergence of data suggest that these prototypic LPS preparations, when highly purified, elicit LPS responses that are restricted in the use of TLR4 as the principal signal-transducing molecule (reviewed in reference 21), which is strongly supported by the finding that synthetic E. coli lipid A activated Toll-like receptor 4 (TLR4) and not TLR2 transfectants (8). However, the lipid A of nonenterobacterial species, e.g., Porphyromonas gingivalis, which has been implicated in the inflammation associated with chronic periodontitis (reviewed in reference 9), differs both structurally and functionally from enterobacterial lipid A. Specifically, the major species of P. gingivalis lipid A is composed of unique branched fatty acids, with longer carbon chains than in enterobacterial lipid A, the absence of a phosphoryl group at position 4Ј of the nonreducing glucosamine, as well as other modifications ( Fig. 1) (1). Consistent with these structural differences is the finding that P. gingivalis LPS activity is poorly inhibited by polymyxin B (12), which has been postulated to inactivate LPS by binding electrostatically to negatively charged phosphate groups, leading to a subsequent interaction of polymyxin B with the hydrophobic fatty acids (25, 33). Although P. gingivalis-induced signaling was shown some time ago to be CD14 dependent (34), site-specific mutagenesis of CD14 suggests that the substitution of certain charged amino acids differentially affects the abilities of Escherichia coli and P. gingivalis LPS to bind CD14 (4, 5). In addition, binding of P. gingivalis LPS to LPS binding protein has been reported to be 100-fo...

Porphyromonas gingivalis is implicated in the etiology of periodontitis. Strains of P. gingivalis have been classified as invasive or noninvasive based on their ability to form abscesses in a mouse model. The purpose of this study was to investigate the ability of P. gingivalis strains to cause abscesses and periodontal bone loss in an experimental rat model and the effect of serum and salivary responses on the pathogenicity of these strains. Subcutaneous injection of animals with P. gingivalis 33277, A7A1-28, W50 or 381 resulted in abscesses in a higher percentage of mice than rats. P. gingivalis 33277 caused lesions at the site of injection, whereas strains A7A1-28 and W50 induced abscesses at distant sites in both mice and rats. Local lesions were seen in rats injected with strain 381, whereas lesions formed distant from the site of injection in mice. When periodontal bone loss was assessed in the experimental rat model, animals challenged with 33277 had the highest amount of horizontal and vertical bone loss. Rats challenged with strain A7A1-28, W50 or 381 had some or no periodontal bone loss compared with controls. Assessment of antibody responses to P. gingivalis in these animals revealed that rats challenged with 33277 had lower levels of serum immunoglobulin G-(IgG) and especially salivary IgA antibody activity than A7A1-28-challenged rats. Serum IgG and in particular salivary IgA anti-P. gingivalis responses were seen in W50- and 381-challenged rats. These results indicate that the ability of P. gingivalis strains to cause abscesses does not relate directly to their periodontal pathogenicity as assessed by periodontal bone loss in the same animal model. The results further suggest the importance of salivary IgA antibody responses in protection against experimental periodontal bone loss after challenge with P. gingivalis.

BACKGROUNDPathogen inactivation (PI) is a new approach to blood safety that may introduce additional costs. This study identifies costs that could be eliminated, thereby mitigating the financial impact.STUDY DESIGN AND METHODSCost information was obtained from five institutions on tests and procedures (e.g., irradiation) currently performed, that could be eliminated. The impact of increased platelet (PLT) availability due to fewer testing losses, earlier entry into inventory, and fewer outdates with a 7-day shelf life were also estimated. Additional estimates include costs associated with managing 1) special requests and 2) test results, 3) quality control and proficiency testing, 4) equipment acquisition and maintenance, 5) replacement of units lost to positive tests, 6) seasonal or geographic testing, and 7) health department interactions.RESULTSAll costs are mean values per apheresis PLT unit in USD ($/unit). The estimated test costs that could be eliminated are $71.76/unit and a decrease in transfusion reactions corresponds to $2.70/unit. Avoiding new tests (e.g., Babesia and dengue) amounts to $41.80/unit. Elimination of irradiation saves $8.50/unit, while decreased outdating with 7-day storage can be amortized to $16.89/unit. Total potential costs saved with PI is $141.65/unit. Costs are influenced by a variety of factors specific to institutions such as testing practices and the location in which such costs are incurred and careful analysis should be performed. Additional benefits, not quantified, include retention of some currently deferred donors and scheduling flexibility due to 7-day storage.CONCLUSIONSWhile PI implementation will result in additional costs, there are also potential offsetting cost reductions, especially after 7-day storage licensing.

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BackgroundBone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture.MethodsA comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared.ResultsPrimary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF.ConclusionsTraditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

BACKGROUND:This pilot assessed transfusion requirements during resuscitation with whole blood followed by standard component therapy (CT) versus CT alone, during a change in practice at a large urban Level I trauma center. METHODS:This was a single-center prospective cohort pilot study. Male trauma patients received up to 4 units of cold-stored low anti-A, anti-B group O whole blood (LTOWB) as initial resuscitation followed by CT as needed (LTOWB + CT). A control group consisting of women and men who presented when LTOWB was unavailable, received CT only (CT group). Exclusion criteria included antiplatelet or anticoagulant medication and death within 24 hours. The primary outcome was total transfusion volume at 24 hours. Secondary outcomes were mortality, morbidity, and intensive care unit-and hospital-free days. RESULTS:Thirty-eight patients received LTOWB, with a median of 2.0 (interquartile range [IQR] 1.0-3.0) units of LTOWB transfused. Thirty-two patients received CT only. At 24 hours after presentation, the LTOWB +CT group had received a median of 2,138 mL (IQR, 1,275-3,325 mL) of all blood products. The median for the CT group was 4,225 mL (IQR, 1,900-5,425 mL; p = 0.06) in unadjusted analysis. When adjusted for Injury Severity Score, sex, and positive Focused Assessment with Sonography for Trauma, LTOWB +CT group patients received 3307 mL of blood products, and CT group patients received 3,260 mL in the first 24 hours (p = 0.95). The adjusted median ratio of plasma to red cells transfused was higher in the LTOWB + CT group (0.85 vs. 0.63 at 24 hours after admission; p = 0.043. Adjusted mortality was 4.4% in the LTOWB + CT group, and 11.7% in the CT group (p = 0.19), with similar complications, intensive care unit-, and hospital-free days in both groups. CONCLUSION:Beginning resuscitation with LTOWB results in equivalent outcomes compared with resuscitation with CT only.

Background Red blood cell (RBC) transfusion is a common medical procedure. While it offers clinical benefits for many, hemodynamically stable patients are often subjected to unwarranted transfusions, with the potential to lead to adverse consequences. We created a real-time clinical decision support (CDS) tool in the electronic health record system to address this problem and optimize transfusion practice as part of an institutional multidisciplinary, team-based patient blood management program. Methods The real-time CDS tool incorporated the transfusion guidelines published by the AABB. The tool was deployed as a dynamic order set within the computerized provider order entry interface. Prior to implementation, extensive education and outreach to increase provider engagement were provided. The CDS tool was launched in September 2015. Results The percentage of guideline-indicated RBC transfusions increased from a baseline of 43.6 to 54.2% while the percentage of multiunit (≥ 2 units) RBC transfusions decreased from 31.3 to 22.7% between September 2014 and July 2019. The estimated minimum cost saving over the entire study period was $36,519.36. Conclusion Our intervention increased guideline-indicated transfusions by 10.6% and reduced multiunit transfusions by 8.6%. The adoption of a dynamic order set for the CDS tool, as opposed to an interruptive alert that displays static alert messages, allowed for more customized and tighter control of RBC orders, leading to a sustained improvement in our transfusion practice.

Background COVID‐19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection, is one of the limited therapeutic options currently available for the treatment of critically ill patients with COVID‐19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion‐transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA‐PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS‐CoV‐2 neutralizing antibodies. Study design and methods This study assessed the impact of A/UVA‐PRT on SARS‐CoV‐2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed. Results A/UVA‐PRT did not negatively impact antibodies to SARS‐CoV‐2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross‐reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses. Conclusion The findings of this study support the selection of effective CCP combined with the use of A/UVA‐PRT in the production of CCP for patients with COVID‐19.