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Objective: The purpose of this study was to determine the M1/M2 macrophage ratio in concentrated bone marrow aspirate (cBMA) in patients undergoing surgical intervention augmented with cBMA for osteochondral lesions of the talus (OLTs)., Design: Samples of peripheral blood (PB), bone marrow aspirate (BMA), and cBMA were collected during the procedure. The samples were analyzed by automated cell counting and multicolor fluorescence-activated cell sorting with specific antibodies recognizing monocytes (CD14+ CD16+) and the M1 (CD86+) and M2 (CD163+CD206+) populations within that monocyte population. Cytokine concentrations within the samples were evaluated with enzyme-linked immunosorbent assay (ELISA). The composition of cBMA was compared between 2 commercially available BMA concentration systems., Results: Thirty-eight patients with a mean age of 43.2 ± 10.1 years old undergoing a surgical procedure for the treatment of OLTs involving the use of cBMA were included. cBMA had a mean fold increase of 4.7 for all white blood cells, 6.1 for monocytes, 7.9 for lymphocytes, 2.4 for neutrophils, and 9.6 for platelets when compared to BMA. The mean M1/M2 ratio for PB, BMA, and cBMA was 15.2 ± 12.0, 20.8 ± 13.3, and 22.1 ± 16.0, respectively. There was a statistically significant higher concentration of interleukin-1 receptor antagonist (IL-1Ra) in the cBMA sample (8243.3 ± 14,837.4 pg/mL) compared to both BMA (3143.0 ± 2218.5 pg/mL) and PB (1847.5 ± 1520.4 pg/mL) samples. The IL-1Ra/IL-1β ratio for PB, BMA, and cBMA was 790.6 ± 581.9, 764.7 ± 675.2, and 235.7 ± 192.1, respectively. There was no difference in the cBMA M1/M2 ratio (19.0 ± 11.1 vs 24.0 ± 18.3) between the Magellan (Isto Biologics, Hopkinton, Massachusetts) and Angel systems (Arthrex Inc, Naples, Florida)., Conclusion: This prospective study found that the M1/M2 ratio in cBMA was 22.1 ± 16.0, with significant patient to patient variation observed. Overall, there was no statistically significant difference in the M1/M2 ratio across PB, BMA, and cBMA samples. This is the first study to characterize the macrophage subpopulation within cBMA, which may have significant clinical implications in future studies., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: J.G.K. is a consultant for Arteriocyte, In2Bones, and Arthrex. J.G.K. receives financial support from the Ohnell Family Foundation, Mr Winston Fischer and Tatiana Rybak.

Aims: To investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization., Methods and Results: Genomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose., Conclusions: Pneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alcorlo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Viruses that carry a positive-sense, single-stranded (+ssRNA) RNA translate their genomes soon after entering the host cell to produce viral proteins, with the exception of retroviruses. A distinguishing feature of retroviruses is reverse transcription, where the +ssRNA genome serves as a template to synthesize a double-stranded DNA copy that subsequently integrates into the host genome. As retroviral RNAs are produced by the host cell transcriptional machinery and are largely indistinguishable from cellular mRNAs, we investigated the potential of incoming retroviral genomes to directly express proteins. Here we show through multiple, complementary methods that retroviral genomes are translated after entry. Our findings challenge the notion that retroviruses require reverse transcription to produce viral proteins. Synthesis of retroviral proteins in the absence of productive infection has significant implications for basic retrovirology, immune responses and gene therapy applications., (© 2024. The Author(s).)

Introduction: Patients with anterior cruciate ligament injury are at high risk of posttraumatic osteoarthritis and their response to reconstructive surgery and rehabilitation vary. Proteins identified in the orchestration of the acute inflammatory response may be predictive of patient outcomes., Objective: An unbiased, bottom-up proteomics approach was used to discover novel targets for therapeutics in relation to dysregulation in the orchestration of inflammatory pathways implicated in persistent joint inflammation subsequent to joint trauma., Methods: Synovial fluid was aspirated from patients at 1 week and 4 weeks after anterior cruciate ligament reconstruction (ACLR) and interleukin 6 (IL-6) concentrations were quantified by enzyme-linked immunosorbent assay. Patients were segregated into IL-6 low and IL-6 high groups based on IL-6 concentrations in synovial fluid at 4-weeks postoperation and proteins in synovial fluid were analyzed using qualitative, bottom-up proteomics. Abundance ratios were calculated for IL-6 high and IL-6 low groups as 4 weeks postoperation:1 week postoperation., Results: A total of 291 proteins were detected in synovial fluid, 34 of which were significantly ( P < .05) differentially regulated between groups. Proteins associated with the classical and alternative complement cascade pathways were increased in the IL-6 high compared to IL-6 low group. Insulin-like growth factor-binding protein 6 (IGFBP-6) was increased by nearly 60-fold in the IL-6 low group., Conclusions: Patients segregated by IL-6 concentration in synovial fluid at 4 weeks post-ACLR demonstrated differential regulation of multiple pathways, providing opportunities to investigate novel targets, such as IGFBP-6, and to take advantage of therapeutics already approved for clinical use in other diseases that target inflammatory pathways, including the complement system., Competing Interests: Declaration of competing interest The author(s) declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.

Anti-inflammatory Regulatory T cells (Tregs) are enriched in the joints of patients with osteoarthritis (OA) compared to healthy joints. Tregs maintain homeostasis through secretion of anti-inflammatory cytokines and cell-to-cell interactions including immune checkpoint signaling. Interleukin-6 (IL-6) is a pleiotropic cytokine secreted by inflamed synoviocytes and chondrocytes that can inhibit or alter Treg function. This study tested the hypothesis that neutralization of IL-6 would enable Treg anti-inflammatory function to resolve inflammation and catabolism elicited by IL-1β in an equine chondrocyte/synoviocyte/Treg tri-culture OA model. Synoviocyte/chondrocyte co-cultures were stimulated with IL-1β, and treated with αIL-6 neutralizing antibody. Activated Tregs secreting IL-10 were added in direct contact with synoviocytes to create a tri-culture. Neutralization of IL-6 partially restored Treg anti-inflammatory functions and, in combination, reduced IL-1β-stimulated synoviocyte MMP13 expression to control levels and restored Acan expression in chondrocytes. IL-6 neutralization alone decreased Il6 expression in chondrocytes and synoviocytes, mitigating IL-6 positive feedback loop. Although Tregs were the primary producers of anti-inflammatory IL-10 and IL-4, they also produced pro-inflammatory IL-17A, as detected by ELIA, which may have been responsible for incomplete rescue of synoviocyte/chondrocyte homeostasis following IL-1β stimulation. Treg secretion of IL-10, IL-4, and IL-17A was not altered by tri-culture conditions or presence of αIL-6, therefore, it was unlikely that Treg phenotype instability occurred. The significant effect of chondrocyte/synoviocyte donor, but not Treg donor, on gene expression and IL-6 concentration in conditioned media, indicated that personalized therapy considering the patient's OA status might be needed for successful implementation of immunotherapy in the context of OA., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Objective and Design: The purpose of this study was to explore pathological processes during the first 4 weeks after anterior cruciate ligament reconstruction (ACLR)., Subjects: Sixteen ACL-injured patients (8 females/8 males, mean age = 19.1, mean BMI = 28.6)., Methods: Arthrocentesis was performed 1 and 4 weeks after ACLR. Proteins in the synovial fluid were identified using nanoLC-ESI-MS/MS. Differentially up- or down-regulated proteins were identified and quantified, and a pathway analysis was performed. All identified proteins were mapped into a protein-protein interaction (PPI) network, and networks of PPIs with a combined score > 0.9 were then visualized., Results: Seven pathways were upregulated after ACLR: PI3K-AKT signaling pathway, extracellular matrix (ECM)-receptor interaction, focal adhesion, protein digestion and absorption, ameobiasis, and platelet activation. Network analyses identified 8 proteins that were differentially upregulated with strong PPI interactions (periostin and 7 collagen-related proteins). Increases in periostin moderately correlated with increases in a synovial fluid biomarker of type II cartilage degradation (ρ = 0.51, p = 0.06)., Conclusion: Pro-inflammatory pathways and periostin were upregulated after ACLR. Periostin demonstrated strong network connections with markers of collagen breakdown, and future work is needed to determine whether periostin may offer a biomarker of early cartilage degradation after ACLR and/or play an active role in early post-traumatic osteoarthritis (PTOA) progression., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Infiltration of cluster of differentiation (CD) 3 + (CD3 + ) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets., Hypothesis: An imbalance of the ratio of regulatory T cells and T helper 17 cells would be associated with disease progression in posttraumatic osteoarthritis, suggesting opportunities for immunomodulatory therapy., Study Design: Descriptive laboratory study., Methods: Synovial fluid was aspirated from the joints of equine clinical patients undergoing arthroscopic surgery for posttraumatic osteoarthritis resulting from intra-articular fragmentation. Joints were classified as having mild or moderate posttraumatic osteoarthritis. Synovial fluid was also obtained from nonoperated horses with normal cartilage. Peripheral blood was obtained from horses with normal cartilage and those with mild and moderate posttraumatic osteoarthritis. Synovial fluid and peripheral blood cells were analyzed by flow cytometry, and native synovial fluid was analyzed by enzyme-linked immunosorbent assay., Results: CD3 + T cells represented 81% of lymphocytes in synovial fluid, which increased in animals with moderate posttraumatic osteoarthritis to 88.3% ( P = .02). CD14 + macrophages were doubled in those with moderate posttraumatic osteoarthritis compared with mild posttraumatic osteoarthritis and controls ( P < .001). Less than 5% of CD3 + T cells found within the joint were forkhead box P3 protein + (Foxp3 + ) regulatory T cells, but a 4- to 8-times higher percentage of nonoperated and mild posttraumatic osteoarthritis joint regulatory T cells secreted interleukin (IL)-10 than peripheral blood Tregs ( P < .005). T regulatory-1 cells that secreted IL-10 but did not express Foxp3 accounted for approximately 5% of CD3 + T cells in all joints. T helper 17 cells and Th17-like regulatory T cells were increased in those with moderate posttraumatic osteoarthritis ( P < .0001) compared with mild and nonoperated patients. IL-10, IL-17A, IL-6, chemokine (C-C motif) ligand (CCL) 2 (CCL2), and CCL5 concentrations detected by enzyme-linked immunosorbent assay in synovial fluid were not different between groups., Conclusions: An imbalance of the ratio of regulatory T cells and T helper 17 cells and an increase in T helper 17 cell-like regulatory T cells in synovial fluid from joints with more severe disease provide novel insights into immunological mechanisms that are associated with posttraumatic osteoarthritis progression and pathogenesis., Clinical Relevance: Early and targeted use of immunotherapeutics in the mitigation of posttraumatic osteoarthritis may improve patient clinical outcomes.

Streptococcus pneumoniae colonizes the human nasopharynx and causes several diseases. Pneumococcal vaccines target the polysaccharide capsule and prevent most serious disease, but there has been an increase in the prevalence of nonencapsulated S. pneumoniae (NESp). Previously, it was thought that a capsule was necessary to cause invasive disease. NESp strains expressing the oligopeptide transporters AliC and AliD have been isolated from patients with invasive disease. The AliC and AliD oligopeptide transporters regulate the expression of several genes, including choline binding protein AC (CbpAC) (a homolog of PspA), which aids in reducing C3b deposition. It is hypothesized that by altering CbpAC expression, AliC and AliD provide protection from classical complement-mediated clearance by reducing C-reactive protein (CRP) binding. Our study demonstrates that AliC and AliD regulate CbpAC expression in NESp and that AliD found in certain serotypes of encapsulated strains regulates PspA expression. C3b deposition was increased in the NESp Δ aliD and encapsulated mutants in comparison to the wild type. NESp strains expressing AliC and AliD have a significant decrease in C1q and CRP deposition in comparison to the Δ aliC Δ aliD mutant. The complement protein C1q is required for NESp clearance in a murine model and increases opsonophagocytosis. By regulating CbpAC expression, NESp inhibits CRP binding to the bacterial surface and blocks classical complement activation, leading to greater systemic survival and virulence. Due to the increase in the prevalence of NESp, it is important to gain a better understanding of NESp virulence mechanisms that aid in establishing disease and persistence within a host by avoiding clearance by the immune system. IMPORTANCE Streptococcus pneumoniae (pneumococcus) can cause a range of diseases. Although there is a robust pneumococcal vaccination program that reduces invasive pneumococcal disease by targeting various polysaccharide capsules, there has been an increase in the isolation of nonvaccine serotypes and nonencapsulated S. pneumoniae (NESp) strains. While most studies of pneumococcal pathogenesis have focused on encapsulated strains, there is little understanding of how NESp causes disease. NESp lacks a protective capsule but contains novel genes, such as aliC and aliD , which have been shown to regulate the expression of numerous genes and to be required for NESp virulence and immune evasion. Furthermore, NESp strains have high transformation efficiencies and harbor resistance to multiple drugs. This could be deleterious to current treatment strategies employed for pneumococcal disease as NESp can be a reservoir of drug resistance genes. Therefore, deciphering how NESp survives within a host and facilitates disease is a necessity that will allow the fabrication of improved, broad-spectrum treatments and preventatives against pneumococcal disease. Our study provides a better understanding of NESp virulence mechanisms during host-pathogen interactions through the examination of genes directly regulated by the NESp proteins AliC and AliD.

Objective: To identify chondroprotective factors as potential disease-modifying osteoarthritis treatments using an unbiased, bottom-up proteomics approach., Samples: Paired equine cartilage explants and synovial membrane were collected postmortem from 4 horses with no history of lameness and grossly normal joints at necropsy., Procedures: Six groups were established: cartilage, synoviocytes, and cartilage + synoviocytes (coculture), all with or without interleukin (IL)-1β. The catabolic effect of IL-1β was verified by glycosaminoglycan (GAG) released from cartilage into media by 1,9-dimethyl-methylene blue assay and cartilage toluidine blue histochemistry. Conditioned media from cocultures with or with IL-1β were submitted for bottom-up proteomic analysis. Synoviocyte gene expression was evaluated using reverse transcription-quantitative PCR (RT-qPCR) for proteins of interest identified in the proteomics scan., Results: GAG content was retained in cartilage when in cocultures treated with IL-1β. Fourteen proteins of interest were selected from the proteomic analysis. From these 14 proteins, metalloproteinase inhibitor 3 precursor (TIMP3), tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), insulin-like growth factor-binding protein 2 (IGFBP2), and alpha-2 macroglobulin (A2M) were selected for synoviocyte gene expression analysis by RT-qPCR. Gene expression of TIMP3 (P = .02) and TNFRSF11B (P = .04) were significantly increased in synoviocytes from cocultures treated with IL-1β compared to controls. Contrary to expectations based on protein expression, IGFBP2 gene expression (P = .04) was significantly decreased in IL-1β-stimulated coculture synoviocytes compared to control coculture synoviocytes. A2M gene expression in synoviocytes was not different between coculture groups., Clinical Relevance: The secretome from synoviocytes could provide a milieu of bioactive factors to restore joint homeostasis in osteoarthritis.

Objective: To gain insight into Treg interactions with synovial tissues in early OA, an equine tri-culture model of OA was used to test the hypothesis that Tregs, in the absence of T Helper 17 ​cells, are sufficient to resolve inflammation elicited by IL-1β., Methods: To model normal and OA joints, synoviocytes were co-cultured with chondrocytes in a transwell system and ± stimulated with IL-1β. Tregs were activated and enriched, then added to co-cultures, creating tri-cultures. At culture end, synoviocytes and chondrocytes were analyzed for gene expression, Treg Foxp3 expression was reexamined by flow cytometry, and conditioned media were evaluated by ELISA., Results: Tregs increased IL-10 and IL-4 in tri-culture media and increased TIMP1 gene expression in synoviocytes and chondrocytes. Tregs increased IL-6 in conditioned media and Il6 gene expression in synoviocytes, which was additive with IL-1β. In chondrocytes, addition of Tregs decreased Col2b gene expression while Acan gene expression was decreased by IL-1β and addition of Tregs. IL-17A was detected in tri-cultures. CCL2 and CCL5 were increased in tri-cultures., Conclusions: In a tri-culture model of OA, addition of Tregs resulted in conditions conducive to chondroprotection including increased concentration of IL-10 and IL-4 in conditioned media and increased gene expression of TIMP1 in both chondrocytes and synoviocytes. However, there was increased concentration of the catabolic cytokine IL-6, and decreased gene expression of Col2b and Acan in IL-1β-stimulated chondrocytes. These results suggest that blocking IL-6 could enhance Treg function in mitigating OA progression., (© 2021 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International (OARSI).)

Background: Birth tissue products from amnion, chorion, umbilical cord, amniotic fluid, or cord blood are frequently marketed as viable sources of stem cells and growth factors. It can be difficult for health care professionals to differentiate implied from explicit conclusions in reported product analyses., Purpose: To provide an educational platform for health care professionals to interpret data presented in the promotion of birth tissue products., Study Design: Descriptive laboratory study and expert opinion; Level of evidence, 5., Methods: A cord blood product was analyzed by 3 methods for cell viability, 2 methods for assessment of cell morphology and cell type, multicolor flow cytometry to identify stem cells, and enzyme-linked immunosorbent assay (ELISA) plus Western blot for analysis of interleukin 1 receptor antagonist protein (IL-1ra). These data were compared with analyses reported by the manufacturer., Results: Cell viability in the cord blood product was less than reported by the manufacturer, the cells were primarily leukocytes, no stem cells were present, and the concentration of IL-1ra was falsely increased due to nonspecific antibody binding in the sample., Conclusion: To assess birth tissue products, health care professionals should consider the following: (1) Understanding fluorescent dyes is important for assessing cell viability data-green does not always mean alive. (2) The report of "cells" in the product does not necessarily mean "stem cells"; microscopic images of at least ×20 or a hemogram should be evaluated to determine cell type (leukocyte, red blood cells, etc). (3) There is no single cluster of differentiation (CD) marker on flow cytometry to identify stem cells. (4) Biological tissues are complex substances, and inaccurately increased measurements of growth factors could be present in ELISA results because most ELISAs are not designed or validated for use in biologics. Furthermore, the reported measurement of growth factors should be considered relative to concentrations in native biological tissues and plasma., Clinical Relevance: Health care professionals should be able to interpret cell viability, cell morphology, stem cell analysis using CD markers, and growth factor analysis when considering use of a birth tissue product in patients.

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae . The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries., Competing Interests: The authors declare no conflict of interest.

Background: Current Streptococcus pneumoniae vaccines selectively target capsular polysaccharide of specific serotypes, leading to an increase in nonencapsulated S. pneumoniae (NESp). Cocolonization by encapsulated pneumococci and NESp increases the opportunity for intraspecies genetic exchange. Acquisition of NESp genes by encapsulated pneumococci could alter virulence and help vaccine-targeted serotypes persist in the host., Methods: Adhesion and invasion assays were performed using immortalized human pharyngeal or lung epithelial cells. In vivo models assessing murine nasopharyngeal colonization and pneumonia, as well as chinchilla otitis media (OM), were also used., Results: Pneumococcal surface protein K (PspK) expression increased encapsulated pneumococcal adhesion and invasion of lung cells and enhanced virulence during pneumonia and OM. Additionally, PspK increased nasopharyngeal colonization, persistence in the lungs, and persistence in the middle ear when expressed in a capsule deletion mutant. Competition experiments demonstrated encapsulated pneumococci expressing PspK also had a selective advantage in both the lungs and nasopharynx., Conclusions: PspK increases pneumococcal virulence during pneumonia and OM. PspK also partially compensates for loss of virulence in the absence of capsule. Additionally, PspK provides a selective advantage in a competitive environment. Therefore, acquisition of PspK increases encapsulated virulence in a condition-dependent manner. Together, these studies demonstrate risks associated with pneumococcal intraspecies genetic exchange.

Nonencapsulated Streptococcus pneumoniae (NESp) is an emerging human pathogen that colonizes the nasopharynx and is associated with noninvasive diseases such as otitis media (OM), conjunctivitis, and nonbacteremic pneumonia. Since capsule expression was previously thought to be necessary for establishment of invasive pneumococcal disease (IPD), serotype-specific polysaccharide capsules are targeted by currently licensed pneumococcal vaccines. Yet, NESp expressing oligopeptide binding proteins AliC and AliD have been isolated during IPD. Thus, we hypothesize AliC and AliD are major NESp virulence determinants that facilitate persistence and development of IPD. Our study reveals that NESp expressing AliC and AliD have intensified virulence compared to isogenic mutants. Specifically, we demonstrate AliC and AliD enhance murine nasopharyngeal colonization and pulmonary infection and are required for OM in a chinchilla model. Furthermore, AliC and AliD increase pneumococcal survival in chinchilla whole blood and aid in resistance to killing by human leukocytes. Comparative proteome analysis revealed significant alterations in protein levels when AliC and AliD were absent. Virulence-associated proteins, including a pneumococcal surface protein C variant (CbpAC), were significantly downregulated, while starvation response indicators were upregulated in the double mutant relative to wild-type levels. We also reveal that differentially expressed CbpAC was essential for NESp adherence to epithelial cells, virulence during OM, reduction of C3b deposition on the NESp surface, and binding to nonspecific IgA. Altogether, the rise in NESp prevalence urges the need to understand how NESp establishes disease and persists in a host. This study highlights the roles of AliC, AliD, and CbpAC in the pathogenesis of NESp. IMPORTANCE Despite the effective, widespread use of licensed pneumococcal vaccines over many decades, pneumococcal infections remain a worldwide burden resulting in high morbidity and mortality. NESp subpopulations are rapidly rising in the wake of capsule-targeted vaccine strategies, yet there is very little knowledge on NESp pathogenic potential and virulence mechanisms. Although NESp lacks a protective capsule, NESp lineages expressing AliC and AliD have been associated with systemic infections. Furthermore, higher antibiotic resistance rates and transformation efficiencies associated with emerging NESp threaten treatment strategies needed to control pneumococcal infections and transmission. Elucidating how NESp survives within a host and establishes disease is necessary for development of broadened pneumococcal prevention methods. Our study identifies virulence determinants and host survival mechanisms employed by NESp with a high pathogenic potential. Moreover, our study also identifies virulence determinants shared by NESp and encapsulated strains that may serve as broad prevention and therapeutic targets., (Copyright © 2018 Bradshaw et al.)

Traditionally regarded as simple foot soldiers of the innate immune response limited to the eradication of pathogens, neutrophils recently emerged as more complex cells endowed with a set of immunoregulatory functions. Using a model of invasive pneumococcal disease, we highlighted an unexpected key role for neutrophils as accessory cells in innate interleukin (IL)-17A production by lung resident Vγ6Vδ1 + T cells via nucleotide-binding oligomerization domain receptor, pyrin-containing 3 (NLRP3) inflammasome-dependent IL-1β secretion. In vivo activation of the NLRP3 inflammasome in neutrophils required both host-derived and bacterial-derived signals. Elaborately, it relies on (i) alveolar macrophage-secreted TNF-α for priming and (ii) subsequent exposure to bacterial pneumolysin for activation. Interestingly, this mechanism can be translated to human neutrophils. Our work revealed the cellular and molecular dynamic events leading to γδT17 cell activation, and highlighted for the first time the existence of a fully functional NLRP3 inflammasome in lung neutrophils. This immune axis thus regulates the development of a protective host response to respiratory bacterial infections.

Streptococcus pneumoniae is commonly found in the human nasopharynx and is the causative agent of multiple diseases. Since invasive pneumococcal infections are associated with encapsulated pneumococci, the capsular polysaccharide is the target of licensed pneumococcal vaccines. However, there is an increasing distribution of non-vaccine serotypes, as well as nonencapsulated S. pneumoniae (NESp). Both encapsulated and nonencapsulated pneumococci possess the polyamine oligo-transport operon (potABCD). Previous research has shown inactivation of the pot operon in encapsulated pneumococci alters protein expression and leads to a significant reduction in pneumococcal murine colonization, but the role of the pot operon in NESp is unknown. Here, we demonstrate deletion of potD from the NESp NCC1 strain MNZ67 does impact expression of the key proteins pneumolysin and PspK, but it does not inhibit murine colonization. Additionally, we show the absence of potD significantly increases biofilm production, both in vitro and in vivo. In a chinchilla model of otitis media (OM), the absence of potD does not significantly affect MNZ67 virulence, but it does significantly reduce the pathogenesis of the virulent encapsulated strain TIGR4 (serotype 4). Deletion of potD also significantly reduced persistence of TIGR4 in the lungs but increased persistence of PIP01 in the lungs. We conclude the pot operon is important for the regulation of protein expression and biofilm formation in both encapsulated and NCC1 nonencapsulated Streptococcus pneumoniae. However, in contrast to encapsulated pneumococcal strains, polyamine acquisition via the pot operon is not required for MNZ67 murine colonization, persistence in the lungs, or full virulence in a model of OM. Therefore, NESp virulence regulation needs to be further established to identify potential NESp therapeutic targets.

Streptococcus pneumoniae infections result in a range of human diseases and are responsible for almost one million deaths annually. Pneumococcal disease is mediated in part through surface structures and an anti-phagocytic capsule. Recent studies have shown that nonencapsulated S. pneumoniae (NESp) make up a significant portion of the pneumococcal population and are able to cause disease. NESp lack some common surface proteins expressed by encapsulated pneumococci, but express surface proteins unique to NESp. A chinchilla model of otitis media (OM) was used to determine the effect various pneumococcal mutations have on pathogenesis in both NESp and encapsulated pneumococci. Epithelial cell adhesion and invasion assays were used to examine the effects in relation to deletion of intrinsic genes or expression of novel genes. A mouse model of colonization was also utilized for comparison of various pneumococcal mutants. It was determined that pneumococcal surface protein K (PspK) and pneumolysin (Ply) affect NESp middle ear pathogenesis, but only PspK affected epithelial cell adhesion. Experiments in an OM model were done with encapsulated strains testing the importance of native virulence factors and treatment of OM. First, a triple deletion of the common virulence factors PspA, PspC, and Ply, (ΔPAC), from an encapsulated background abolished virulence in an OM model while a PspC mutant had detectable, but reduced amounts of recoverable bacteria compared to wildtype. Next, treatment of OM was effective when starting antibiotic treatment within 24 h with resolution by 48 h post-treatment. Expression of NESp-specific virulence factor PspK in an encapsulated strain has not been previously studied, and we showed significantly increased adhesion and invasion of human epithelial cells by pneumococci. Murine colonization was not significantly increased when an encapsulated strain expressed PspK, but colonization was increased when a capsule mutant expressed PspK. The ability of PspK expression to increase colonization in a capsule mutant despite no increase in adhesion can be attributed to other functions of PspK, such as sIgA binding or immune modulation. OM is a substantial economic burden, thus a better understanding of both encapsulated pneumococcal pathogenesis and the emerging pathogen NESp is necessary for effective prevention and treatment.

Streptococcus pneumoniae is an important human pathogen. To cause disease, it must first colonize the nasopharynx. The widespread use of pneumococcal-conjugate vaccines which target the capsular polysaccharide has led to decreased nasopharyngeal carriage of vaccine serotypes, but a concomitant increase in carriage of non-vaccine serotypes and nonencapsulated S. pneumoniae (NESp). Some NESp express pneumococcal surface protein K (PspK), a virulence factor shown to contribute to nasopharyngeal colonization. We present the case of a child with chronic adenoiditis caused by a PspK(+) NESp. We tested the pneumococcal isolate, designated C144.66, for antimicrobial resistance, the presence of the pspK gene and the expression of PspK. Sequence typing and genome sequencing were performed. C144.66 was found to be resistant to erythromycin and displayed intermediate resistance to penicillin and trimethoprim/sulfamethoxazole. C144.66 has the pspK gene in place of the capsule locus. Additionally, PspK expression was confirmed by flow cytometry. NESp are a growing concern as an emerging human pathogen, as current pneumococcal vaccines do not confer immunity against them. An inability to vaccinate against NESp may result in increased carriage and associated pathology.

While significant protection from pneumococcal disease has been achieved by the use of polysaccharide and polysaccharide-protein conjugate vaccines, capsule-independent protection has been limited by serotype replacement along with disease caused by nonencapsulatedStreptococcus pneumoniae(NESp). NESp strains compose approximately 3% to 19% of asymptomatic carriage isolates and harbor multiple antibiotic resistance genes. Surface proteins unique to NESp enhance colonization and virulence despite the lack of a capsule even though the capsule has been thought to be required for pneumococcal pathogenesis. Genes for pneumococcal surface proteins replace the capsular polysaccharide (cps) locus in some NESp isolates, and these proteins aid in pneumococcal colonization and otitis media (OM). NESp strains have been isolated from patients with invasive and noninvasive pneumococcal disease, but noninvasive diseases, specifically, conjunctivitis (85%) and OM (8%), are of higher prevalence. Conjunctival strains are commonly of the so-called classical NESp lineages defined by multilocus sequence types (STs) ST344 and ST448, while sporadic NESp lineages such as ST1106 are more commonly isolated from patients with other diseases. Interestingly, sporadic lineages have significantly higher rates of recombination than classical lineages. Higher rates of recombination can lead to increased acquisition of antibiotic resistance and virulence factors, increasing the risk of disease and hindering treatment. NESp strains are a significant proportion of the pneumococcal population, can cause disease, and may be increasing in prevalence in the population due to effects on the pneumococcal niche caused by pneumococcal vaccines. Current vaccines are ineffective against NESp, and further research is necessary to develop vaccines effective against both encapsulated and nonencapsulated pneumococci., (Copyright © 2016 Keller et al.)

Across bacterial species, metal binding proteins can serve functions in pathogenesis in addition to regulating metal homeostasis. We have compared and contrasted the activities of zinc (Zn2+)-binding lipoproteins AdcA and AdcAII in the Streptococcus pneumoniae TIGR4 background. Exposure to Zn2+-limiting conditions resulted in delayed growth in a strain lacking AdcAII (ΔAdcAII) when compared to wild type bacteria or a mutant lacking AdcA (ΔAdcA). AdcAII failed to interact with the extracellular matrix protein laminin despite homology to laminin-binding proteins of related streptococci. Deletion of AdcA or AdcAII led to significantly increased invasion of A549 human lung epithelial cells and a trend toward increased invasion in vivo. Loss of AdcAII, but not AdcA, was shown to negatively impact early colonization of the nasopharynx. Our findings suggest that expression of AdcAII affects invasiveness of S. pneumoniae in response to available Zn2+ concentrations.

Current vaccinations are effective against encapsulated strains of Streptococcus pneumoniae, but they do not protect against nonencapsulated Streptococcus pneumoniae (NESp), which is increasing in colonization and incidence of pneumococcal disease. Vaccination with pneumococcal proteins has been assessed for its ability to protect against pneumococcal disease, but several of these proteins are not expressed by NESp. Pneumococcal surface protein K (PspK), an NESp virulence factor, has not been assessed for immunogenic potential or host modulatory effects. Mammalian cytokine expression was determined in an in vivo mouse model and in an in vitro cell culture system. Systemic and mucosal mouse immunization studies were performed to determine the immunogenic potential of PspK. Murine serum and saliva were collected to quantitate specific antibody isotype responses and the ability of antibody and various proteins to inhibit epithelial cell adhesion. Host cytokine response was not reduced by PspK. NESp was able to colonize the mouse nasopharynx as effectively as encapsulated pneumococci. Systemic and mucosal immunization provided protection from colonization by PspK-positive (PspK(+)) NESp. Anti-PspK antibodies were recovered from immunized mice and significantly reduced the ability of NESp to adhere to human epithelial cells. A protein-based pneumococcal vaccine is needed to provide broad protection against encapsulated and nonencapsulated pneumococci in an era of increasing antibiotic resistance and vaccine escape mutants. We demonstrate that PspK may serve as an NESp target for next-generation pneumococcal vaccines. Immunization with PspK protected against pneumococcal colonization, which is requisite for pneumococcal disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Use of the pneumococcal conjugate vaccine has led to serotype replacement of carriage and acute otitis media (AOM) pneumococcal isolates. Increases in nonencapsulated Streptococcus pneumoniae (NESp) isolates have also occurred, and there are increasing reports of NESp-associated disease. Disease prevalence and virulence factors of NESp isolates have not been studied., Methods: A chinchilla model of pneumococcal AOM was utilized, and disease was assessed through bacterial enumeration along with scoring visible signs of pathology. An adhesion-invasion assay using a human epithelial cell line was performed., Results: Nonencapsulated Streptococcus pneumoniae strains containing pneumococcal surface protein K (PspK) were more likely to cause AOM and pathology upon infection. Deletion of PspK from an isolate significantly reduced bacterial loads. Increased epithelial cell adhesion correlated with increased virulence of NESp isolates naturally lacking PspK. Furthermore, expression of PspK by an avirulent NESp resulted in virulence., Conclusions: The presence of PspK increased the disease potential of NESp. Pneumococcal surface protein K is not the only virulence factor of NESp in AOM. Expression of PspK in an avirulent NESp mediated the progression to pneumococcal disease. Genetic exchange between pneumococci may allow dissemination of PspK, increasing the potential of NESp disease. The current study is the first report of a NESp-specific virulence factor.

Nonencapsulated Streptococcus pneumoniae can colonize the human nasopharynx and cause conjunctivitis and otitis media. Different deletions in the capsular polysaccharide biosynthesis locus and different multilocus sequence types have been described for nonencapsulated strains. Draft genome sequences were generated to provide insight into the genomic diversity of these strains.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeable S. pneumoniae (NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K (pspK), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.

A psychoanalytic model of preschool consultation is presented, born of the conviction that psychoanalysis has powerful tools with which to tackle some of our most difficult and entrenched community problems. Since trauma is known to impact our ability to think reflectively and symbolically (Fonagy, Moran, and Target 1993), the clinical methods of psychoanalysis-drawing on the ideas of container/contained, the capacity to hold alternate points of view, and the capacity to reflect empathically (here with teachers in the face of their at times harsh and insensitive treatment of students)-are crucial to success in work with cumulatively traumatized staff (Khan 1963). The strongest resistances to consultation arise from an anti-attachment system developed as a result of cumulative trauma and operating at multiple levels in the preschool. Consultation services are organized around an understanding of this anti-attachment system. At various levels of the system, including staff, parents, and children, consultants work to facilitate secure relationships in the preschool setting. These relationships foster recognition of children's emotional complexities and build cooperative links between staff and parents, in an atmosphere that otherwise might often push adults into fearful, self-protective states that interfere with their contact with children's states of mind.

Violence against women by their male partners is widespread and infrequently identified as a causal factor in multiple physical and psychological problems of female patients in medical and psychiatric settings. Three main countertransferences that interfere with accurate identification of battered women are described: (1) counter- identification, (2)countertransference rage, and (3) countertransference helplessness. Battering men and battered women are found in all levels of society, although younger, lower income, less-educated men who have observed parental violence in their own home are at higher risk of abusing their spouses. Additionally, antisocial personality disorder, depression, and/or alcohol and drug abuse increase the risk of male violence in the home. Contrary to popular belief, the husband-to-wife violence is usually motivated by his need to control her rather than a result of his loss of control. Battered women show no consistent prebattering risk markers, except for a history of parental violence in their family of origin. Violence against women by their male partners is a serious public health problem that has not been adequately addressed by the medical and psychiatric professions. Myths and clinical realities of battered women are described and detailed recommendations for clinical inquiry and evaluation of level of danger are given.

Psychoanalytic treatment of addiction is often ineffective because therapists fail to recognize addiction as a discrete disorder. The author reviews psychoanalytic theories of addiction and presents an alternative concept comprising biological, behavioral, and psychological characteristics. She compares the structural similarities between addiction and perversion and describes the use of the addictive substance as a fetish object. Finally, she discusses the implications of psychoanalytically oriented treatment and advocates a multimodal treatment approach.