Emerging concepts in the pathogenesis of the Streptococcus pneumoniae: From nasopharyngeal colonizer to intracellular pathogen.

Streptococcus pneumoniae (the pneumococcus) is a human respiratory tract pathogen and a major cause of morbidity and mortality globally. Although the pneumococcus is a commensal bacterium that colonizes the nasopharynx, it also causes lethal diseases such as meningitis, sepsis, and pneumonia, especially in immunocompromised patients, in the elderly, and in young children. Due to the acquisition of antibiotic resistance and the emergence of nonvaccine serotypes, the pneumococcus has been classified as one of the priority pathogens for which new antibacterials are urgently required by the World Health Organization, 2017. Understanding molecular mechanisms behind the pathogenesis of pneumococcal infections and bacterial interactions within the host is crucial to developing novel therapeutics. Previously considered to be an extracellular pathogen, it is becoming evident that pneumococci may also occasionally establish intracellular niches within the body to escape immune surveillance and spread within the host. Intracellular survival within host cells also enables pneumococci to resist many antibiotics. Within the host cell, the bacteria exist in unique vacuoles, thereby avoiding degradation by the acidic lysosomes, and modulate the expression of its virulence genes to adapt to the intracellular environment. To invade and survive intracellularly, the pneumococcus utilizes a combination of virulence factors such as pneumolysin (PLY), pneumococcal surface protein A (PspA), pneumococcal adhesion and virulence protein B (PavB), the pilus-1 adhesin RrgA, pyruvate oxidase (SpxB), and metalloprotease (ZmpB). In this review, we discuss recent findings showing the intracellular persistence of Streptococcus pneumoniae and its underlying mechanisms.

CD163 and IgG codefend against cytotoxic hemoglobin via autocrine and paracrine mechanisms.

Lysis of RBCs during numerous clinical settings such as severe hemolytic anemia, infection, tissue injury, or blood transfusion releases the endogenous damage-associated molecular pattern, hemoglobin (Hb), into the plasma. The redox-reactive Hb generates cytotoxic reactive oxygen species, disrupting the redox balance and impairing the immune-responsive blood cells. Therefore, it is crucial to understand how the immune system defends against the cytotoxic Hb. We identified a shortcut "capture and quench" mechanism of detoxification of Hb by the monocyte scavenger receptor CD163, independent of the well-known dominant antioxidant, haptoglobin. Our findings support a highly efficient two-pass mechanism of detoxification and clearance of Hb: 1) a direct suppression of Hb-pseudoperoxidase activity by CD163, involving an autocrine loop of CD163 shedding, sequestration of Hb, recycling, and homeostasis of CD163 in human monocytes and 2) paracrine transactivation of endothelial cells by the shedded soluble CD163 (sCD163), which further detoxifies and clears residual Hb. We showed that sCD163 and IgG interact with free Hb in the plasma and subsequently the sCD163-Hb-IgG complex is endocytosed into monocytes via FcγR. The endocytosed sCD163 is recycled to restore the homeostasis of CD163 on the monocyte membrane in an autocrine cycle, whereas the internalized Hb is catabolized. Using ex vivo coculture experiments, we demonstrated that the monocyte-derived sCD163 and IgG shuttle residual plasma Hb into the proximal endothelial cells. These findings suggest that CD163 and IgG collaborate to engage monocytes and endothelial cells in a two-pass detoxification mechanism to mount a systemic defense against Hb-induced oxidative stress.

Chondroblastoma of the talus.

We report the case of a 13-year-old male child who presented with a painful left ankle. On imaging (radiography and computed tomography scan with 3-dimensional reconstruction views), an osteolytic lesion in the body of the talus was revealed. Open biopsy, curettage, and fibular bone grafting were done, and the specimen was sent for histopathologic examination. The histopathologic report confirmed the specimen to be chondroblastoma of the talus bone. Chondroblastoma is a rare benign cartilaginous neoplasm that accounts for approximately 1% of all bone tumors and characteristically arises in the epiphysis of a long bone, particularly the humerus, tibia, and femur. Chondroblastoma can affect people of all ages. It is, however, most common in children and young adults aged 10 to 20 years. Chondroblastoma in a tarsal bone is a rare entity. Managing chondroblastoma of the talus with curettage and bone grafting has shown good outcomes.

Emerging Roles of Extracellular Vesicles in Pneumococcal Infections: Immunomodulators to Potential Novel Vaccine Candidates.

The Gram-positive bacterial pathogen, Streptococcus pneumoniae is a major global health threat that kills over one million people worldwide. The pneumococcus commonly colonizes the nasopharynx asymptomatically as a commensal, but is also capable of causing a wide range of life-threatening diseases such as pneumonia, meningitis and septicemia upon migration into the lower respiratory tract and spread to internal organs. Emergence of antibiotic resistant strains and non-vaccine serotypes has led to the classification of pneumococcal bacteria as a priority pathogen by the World Health Organization that needs urgent research into bacterial pathogenesis and development of novel vaccine strategies. Extracellular vesicles are spherical membrane bound structures that are released by both pathogen and host cells, and influence bacterial pathogenesis as well as the immune response. Recent studies have found that while bacterial vesicles shuttle virulence factors and toxins into host cells and regulate inflammatory responses, vesicles released from the infected host cells contain both bacterial and host proteins that are antigenic and immunomodulatory. Bacterial membrane vesicles have great potential to be developed as cell-free vaccine candidates in the future due to their immunogenicity and biostability. Host-derived vesicles isolated from patient biofluids such as blood and bronchoalveolar lavage could be used to identify potential diagnostic biomarkers as well as engineered to deliver desired payloads to specific target cells for immunotherapy. In this review, we summarize the recent developments on the role of bacterial and host vesicles in pneumococcal infections and future prospects in developing novel therapeutics and diagnostics for control of invasive pneumococcal diseases.

Quantitative susceptibility mapping of both ring and non-ring white matter lesions in relapsing remitting multiple sclerosis.

BACKGROUND: Recent evidence suggests that the presence of magnetic susceptibility changes in MS lesions correlates with patients' expanded disability status scale (EDSS). PURPOSE: This study evaluated the presence of ring lesions (RLs) and non-RLs, as well as changes in lesion susceptibility and lesion volume over a two-year time period in relapsing-remitting multiple sclerosis (RRMS) patients and compared these measures to the EDSS. STUDY TYPE: Longitudinal cohort. MATERIALS AND METHODS: A total of forty-three (43) patients with RRMS were recruited for this study. All subjects underwent 3 T MRI at baseline and forty-one (41) subjects had follow-up scans over a two-year period. The protocol included T2 fluid attenuated inversion recovery (FLAIR), pre- and post-contrast 2D T1-weighted gradient echo imaging and susceptibility weighted imaging (SWI) sequences. All data were acquired prior to gadolinium-based contrast agent injection except for SWI and post-contrast T1-weighted data. Mean and peak susceptibilities of lesions were measured using quantitative susceptibility mapping (QSM). For the RLs, inner-/outer-diameters from SWI data and absolute intensity from the FLAIR data were measured. The susceptibility and volumes of RLs and non-RLs were correlated with EDSS. RESULTS: The number of RLs correlated positively with EDSS (p-value = 0.04), but the RL volumes did not show a correlation with EDSS. Measurements of the annular ring and regions with high susceptibility remained constant over time (p = 0.2). For non-ring QSM-positive (QSM+) lesions, the average susceptibility was significantly correlated to EDSS (p < 0.01.) This was also the case for lesion volume, as well as the product of volume and susceptibility when compared to EDSS (p < 0.01 for both). CONCLUSIONS: The susceptibility distribution and lesion volumes for the RLs remained almost constant over time, suggesting that the changes in pathophysiology of the RLs is a gradual process. On the other hand, the presence of more than one RL along with both volume and susceptibility of non-RL QSM+ lesions were significantly associated with increased disability as measured by EDSS. These findings may strengthen the role of QSM in assessing MS patients radiologically.

Same Day versus Overnight Discharge in Patients Undergoing Ablation for Atrial Fibrillation (SODA) Study.

Patients undergoing catheter ablation for atrial fibrillation (AF) are routinely admitted for observation overnight in the hospital. With the rising incidence of AF among the population, increased volumes of procedures are placing increased demands on hospital resources. The purpose of this study was to evaluate the efficacy and safety of same day discharge in patients undergoing ablation for AF when compared to patients admitted for overnight observation. We performed a retrospective analysis using a multicenter cohort among patients who were discharged home after elective pulmonary vein isolation (PVI) ablation for AF. In our analysis, we found no statistically significant difference between patients discharged on the day of their procedure when compared to patients admitted for overnight observation in terms of 90-day readmission, major adverse cardiovascular events and death. This study shows that same day discharge after AF ablation is a feasible option. Future studies are needed to elicit the appropriate protocol to use.

A Comparison of Magnetic Resonance Imaging Methods to Assess Multiple Sclerosis Lesions: Implications for Patient Characterization and Clinical Trial Design.

Magnetic resonance imaging (MRI) is a sensitive imaging modality for identifying inflammatory and/or demyelinating lesions, which is critical for a clinical diagnosis of MS and evaluating drug responses. There are many unique means of probing brain tissue status, including conventional T1 and T2 weighted imaging (T1WI, T2WI), T2 fluid attenuated inversion recovery (FLAIR), magnetization transfer, myelin water fraction, diffusion tensor imaging (DTI), phase-sensitive inversion recovery and susceptibility weighted imaging (SWI), but no study has combined all of these modalities into a single well-controlled investigation. The goals of this study were to: compare different MRI measures for lesion visualization and quantification; evaluate the repeatability of various imaging methods in healthy controls; compare quantitative susceptibility mapping (QSM) with myelin water fraction; measure short-term longitudinal changes in the white matter of MS patients and map out the tissue properties of the white matter hyperintensities using STAGE (strategically acquired gradient echo imaging). Additionally, the outcomes of this study were anticipated to aid in the choice of an efficient imaging protocol reducing redundancy of information and alleviating patient burden. Of all the sequences used, T2 FLAIR and T2WI showed the most lesions. To differentiate the putative demyelinating lesions from inflammatory lesions, the fusion of SWI and T2 FLAIR was used. Our study suggests that a practical and efficient imaging protocol combining T2 FLAIR, T1WI and STAGE (with SWI and QSM) can be used to rapidly image MS patients to both find lesions and study the demyelinating and inflammatory characteristics of the lesions.

Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease.

Cholesterol-dependent cytolysins (CDCs) are essential virulence factors for many human pathogens like Streptococcus pneumoniae (pneumolysin, PLY), Streptococcus pyogenes (streptolysin O, SLO), and Listeria monocytogenes (Listeriolysin, LLO) and induce cytolysis and inflammation. Recently, we identified that pneumococcal PLY interacts with the mannose receptor (MRC-1) on specific immune cells thereby evoking an anti-inflammatory response at sublytic doses. Here, we identified the interaction sites between MRC-1 and CDCs using computational docking. We designed peptides from the CTLD4 domain of MRC-1 that binds to PLY, SLO, and LLO, respectively. In vitro, the peptides blocked CDC-induced cytolysis and inflammatory cytokine production by human macrophages. Also, they reduced PLY-induced damage of the epithelial barrier integrity as well as blocked bacterial invasion into the epithelium in a 3D lung tissue model. Pre-treatment of human DCs with peptides blocked bacterial uptake via MRC-1 and reduced intracellular bacterial survival by targeting bacteria to autophagosomes. In order to use the peptides for treatment in vivo, we developed calcium phosphate nanoparticles (CaP NPs) as peptide nanocarriers for intranasal delivery of peptides and enhanced bioactivity. Co-administration of peptide-loaded CaP NPs during infection improved survival and bacterial clearance in both zebrafish and mice models of pneumococcal infection. We suggest that MRC-1 peptides can be employed as adjunctive therapeutics with antibiotics to treat bacterial infections by countering the action of CDCs.

Genomic Characterization of the Emerging Pathogen Streptococcus pseudopneumoniae.

Streptococcus pseudopneumoniae is a close relative of the major human pathogen S. pneumoniae It is increasingly associated with lower-respiratory-tract infections (LRTI) and a high prevalence of antimicrobial resistance (AMR). S. pseudopneumoniae is difficult to identify using traditional typing methods due to similarities with S. pneumoniae and other members of the mitis group (SMG). Using whole-genome sequencing of LRTI isolates and a comparative genomic approach, we found that a large number of pneumococcal virulence and colonization genes are present in the core S. pseudopneumoniae genome. We also reveal an impressive number of novel surface-exposed proteins encoded by the genome of this species. In addition, we propose a new and entirely specific molecular marker useful for the identification of S. pseudopneumoniae Phylogenetic analyses of S. pseudopneumoniae show that specific clades are associated with allelic variants of core proteins. Resistance to tetracycline and macrolides, the two most common types of resistance, were found to be encoded by Tn916-like integrating conjugative elements and Mega-2. Overall, we found a tight association of genotypic determinants of AMR and phenotypic AMR with a specific lineage of S. pseudopneumoniae Taken together, our results shed light on the distribution in S. pseudopneumoniae of genes known to be important during invasive disease and colonization and provide insight into features that could contribute to virulence, colonization, and adaptation.IMPORTANCES. pseudopneumoniae is an overlooked pathogen emerging as the causative agent of lower-respiratory-tract infections and associated with chronic obstructive pulmonary disease (COPD) and exacerbation of COPD. However, much remains unknown on its clinical importance and epidemiology, mainly due to the lack of specific markers to distinguish it from S. pneumoniae Here, we provide a new molecular marker entirely specific for S. pseudopneumoniae and offer a comprehensive view of the virulence and colonization genes found in this species. Finally, our results pave the way for further studies aiming at understanding the pathogenesis and epidemiology of S. pseudopneumoniae.

Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival.

Streptococcus pneumoniae (the pneumococcus) is a major cause of mortality and morbidity globally, and the leading cause of death in children under 5 years old. The pneumococcal cytolysin pneumolysin (PLY) is a major virulence determinant known to induce pore-dependent pro-inflammatory responses. These inflammatory responses are driven by PLY-host cell membrane cholesterol interactions, but binding to a host cell receptor has not been previously demonstrated. Here, we discovered a receptor for PLY, whereby pro-inflammatory cytokine responses and Toll-like receptor signalling are inhibited following PLY binding to the mannose receptor C type 1 (MRC-1) in human dendritic cells and mouse alveolar macrophages. The cytokine suppressor SOCS1 is also upregulated. Moreover, PLY-MRC-1 interactions mediate pneumococcal internalization into non-lysosomal compartments and polarize naive T cells into an interferon-γlow, interleukin-4high and FoxP3+ immunoregulatory phenotype. In mice, PLY-expressing pneumococci colocalize with MRC-1 in alveolar macrophages, induce lower pro-inflammatory cytokine responses and reduce neutrophil infiltration compared with a PLY mutant. In vivo, reduced bacterial loads occur in the airways of MRC-1-deficient mice and in mice in which MRC-1 is inhibited using blocking antibodies. In conclusion, we show that pneumococci use PLY-MRC-1 interactions to downregulate inflammation and enhance bacterial survival in the airways. These findings have important implications for future vaccine design.

Vitamin D Promotes Pneumococcal Killing and Modulates Inflammatory Responses in Primary Human Neutrophils.

Streptococcus pneumoniae is a major human pathogen and a leading cause of pneumonia, septicemia, and meningitis worldwide. Despite clinical studies linking vitamin D deficiency and pneumonia, molecular mechanisms behind these observations remain unclear. In particular, the effects of vitamin D on neutrophil responses remain unknown. Using pneumococcal strains, primary neutrophils isolated from human blood, and sera from patients with frequent respiratory tract infections (RTIs), we investigated the effects of vitamin D on neutrophil bactericidal and inflammatory responses, including pattern recognition receptors, antimicrobial peptides, and cytokine regulation. We found that vitamin D upregulated pattern recognition receptors, TLR2, and NOD2, and induced the antimicrobial human neutrophil peptides (HNP1-3) and LL-37, resulting in increased killing of pneumococci in a vitamin D receptor-dependent manner. Antibodies targeting HNP1-3 inhibited bacterial killing. Vitamin D supplementation of serum from patients with bacterial RTIs enhanced neutrophil killing. Moreover, vitamin D lowered inflammatory cytokine production by infected neutrophils via IL-4 production and the induction of suppressor of cytokine signaling (SOCS) proteins SOCS-1 and SOCS-3, leading to the suppression of NF-κB signaling. Thus, vitamin D enhances neutrophil killing of S. pneumoniae while dampening excessive inflammatory responses and apoptosis, suggesting that vitamin D could be used alongside antibiotics when treating pneumococcal infections.

Perilunate Dislocation - Case Report and Review of Literature.

Perilunate dislocations are rare injuries comprising of less than 10% of all wrist injuries. These usually occur after high-energy trauma to the wrist. One-fourth (25%) of perilunate dislocations are missed at the initial presentation. We report a case of perilunate dislocation in a patient who presented late, about two months after trauma. He underwent open reduction through a single dorsal approach and internal fixation with K-wires. Aggressive physiotherapy exercises were started after removal of K-wires. He regained near normal full range of motions at the wrist by the end of four months and showed no recurrence of the dislocation. In conclusion, perilunate dislocations should be considered as one of the important differential diagnosis in patients with history of high-energy trauma to the wrist. Early diagnosis and treatment is necessary to prevent the potential risk of avascular necrosis of lunate and scaphoid and secondary osteoarthritis. The best results can be obtained with open reduction and internal fixation with K-wires via a single dorsal approach.

Cinobufagin Modulates Human Innate Immune Responses and Triggers Antibacterial Activity.

The traditional Chinese medicine Chan-Su is widely used for treatment of cancer and cardiovascular diseases, but also as a remedy for infections such as furunculosis, tonsillitis and acute pharyngitis. The clinical use of Chan-Su suggests that it has anti-infective effects, however, the mechanism of action is incompletely understood. In particular, the effect on the human immune system is poorly defined. Here, we describe previously unrecognized immunomodulatory activities of cinobufagin (CBG), a major bioactive component of Chan-Su. Using human monocyte-derived dendritic cells (DCs), we show that LPS-induced maturation and production of a number of cytokines was potently inhibited by CBG, which also had a pro-apoptotic effect, associated with activation of caspase-3. Interestingly, CBG triggered caspase-1 activation and significantly enhanced IL-1ß production in LPS-stimulated cells. Finally, we demonstrate that CBG upregulates gene expression of the antimicrobial peptides (AMPs) hBD-2 and hBD-3 in DCs, and induces secretion of HNP1-3 and hCAP-18/LL-37 from neutrophils, potentiating neutrophil antibacterial activity. Taken together, our data indicate that CBG modulates the inflammatory phenotype of DCs in response to LPS, and triggers an antibacterial innate immune response, thus proposing possible mechanisms for the clinical effects of Chan-Su in anti-infective therapy.

Preferential silent survival of intracellular bacteria in hemoglobin-primed macrophages.

Hemolysis releases hemoglobin (Hb), a prooxidant, into circulation. While the heme iron is a nutrient for the invading pathogens, it releases ROS, which is both microbicidal and cytotoxic, making it a double-edged sword. Previously, we found a two-pass detoxification mechanism involving the endocytosis of Hb into monocytes in collaboration with vascular endothelial cells to overcome oxidative damage. This prompted us to examine the effect of Hb priming on host cell viability and intracellular bacterial clearance during a hemolytic infection. Here, we demonstrate that Hb-primed macrophages harbor a higher intracellular bacterial load but with suppressed apoptosis. p-ERK and p-p38 MAPK were significantly downregulated, with concomitant impairment of Bax and downstream caspases. The Hb-primed cells harboring intracellular bacteria upregulated anti-inflammatory IL-10 and downregulated proinflammatory TNF-α, which further enhanced the infectivity of the neighboring cells. Our findings suggest that opportunistic intracellular pathogens exploit the Hb-scavenging machinery of the host to silently persist within the circulating phagocytes by suppressing apoptosis while escaping immune surveillance.