bluuebunny/crossref_metadata_2025_split huggingface dataset:
252. Alveolar Macrophages Modulate Host Inflammatory Responses to Pulmonary Melioidosis
Authors: Baker Sarah M , West Timothy E 
Published: November 2023

Abstract

Background
Melioidosis is an emerging infection caused by the facultatively intracellular pathogen and Tier 1 select agent Burkholderia pseudomallei (Bps). Pneumonia, the most common manifestation of melioidosis, predicts poor prognosis, yet the pulmonary immune response to Bps is poorly understood. Alveolar macrophages (AMs) are innate immune cells in the lung and are distinct from monocytes and other macrophages in their response to pathogens, yet their role in host defense against melioidosis is unknown. We sought to determine if AMs modulate bacterial replication, dissemination, pulmonary inflammatory response, and health outcomes to Burkholderia using a murine model of melioidosis.

Methods
To deplete mice of AMs, we instilled 100μl clodronate-liposomes or PBS-liposomes intratracheally into C57Bl/6 mice and confirmed AM depletion by flow cytometry. Twenty-four hours after AM depletion, we infected mice by aerosolization with a sub-lethal dose of 7.8 x 103 CFU/lung of B. thailandensis (Bt), a BSL-2-compliant surrogate model of Bps infection. At 24 and 48 hours after infection we quantified pulmonary bacterial burdens, bacterial dissemination to the spleen, lung homogenate cytokines, and clinical markers of illness (health score, weight, and temperature) in mice depleted of AMs and compared these to sham-depleted mice.

Results
Mice deplete of AMs had fewer bacteria in the lungs 24 hours after infection and similar numbers of bacteria at 48 hours; there were no differences in bacterial dissemination or clinical health outcomes. Mice deplete of AMs produced less of the inflammasome-associated cytokines IL-18 and IL-1β at 24 and 48 hours and produced less of the pro-inflammatory cytokine TNF-α
and the neutrophil chemokine MIP-2 at 24 hours. When adjusting for lung bacterial CFUs, mice deplete of AMs still produced less IL-1β at 24 hours.

Conclusion
In a murine model of sub-lethal pulmonary melioidosis, AMs do not contribute to early bacterial restriction and may instead act as an intracellular Burkholderia replication niche or otherwise facilitate replication in the lung. However, AMs are likely to be a main source of IL-1β in the lung, signifying their relevance in the canonical inflammasome pathways implicated in host response melioidosis.

Disclosures
All Authors: No reported disclosures
262. Inhibiting macrophage Warburg effect blocks DNA hyper-methylation and restores immune function
Authors: Abhimanyu Abhimanyu , Ladki Malik , Sheikh Daanish , Nishiguchi Tomoki , Carrero Longlax Santiago , DiNardo Andrew 
Published: November 2023

Abstract

Background
Severe and chronic infections, such as sepsis and Tuberculosis (TB), lead to epigenetic-mediated immune suppression. Cancer studies have demonstrated that perturbations in TCA cycle intracellular metabolites trigger global changes in the epigenetic landscape, resulting in immune suppression that persists after microbial eradication. Therefore inhibiting the TCA cycle may mitigate epigenetic-mediated immune suppression.

Methods
We applied complementary approaches, including data mining public tuberculosis and sepsis datasets, an in vitro LPS-stimulated macrophage tolerance model, DNA methylation (Methyl EPIC), confocal microscopy, and reverse phase protein array (RPPA) to investigate mechanisms of post-infectious immune suppression. Results were validated in tuberculosis patients in a clinical trial using an mTOR inhibitor.

Results
Patients with TB and sepsis both demonstrated increased expression of genes involved in glycolysis and the tricarboxylic acid (TCA) cycle, which correlated with an increase in DNA methylation. Human monocytes stimulated with LPS in vitro developed immune tolerance that correlated with DNA hypermethylation. LPS induced the TCA enzymes isocitrate dehydrogenase, citrate synthetase, as well as DNMT3b to translocate into the nucleus. Inhibition of TCA cycle overactivation blocked LPS-induced immune suppression and DNA hyper-methylation. Phase 2 TCA metabolites succinate and itaconate mimicked LPS-induced immune suppression and DNA hyper-methylation. In vitro studies were validated in vivo with TB patients receiving mTOR inhibition (everolimus) having less DNA hyper-methylation.

Conclusion
TCA cycle activation regulates DNA methylation and immune tolerance. These in vitro and in vivo studies demonstrate that inhibiting the TCA cycle can mitigate epigenetic-mediated immune silencing, potentially identifying a means to prevent post-infectious immune suppression.

Disclosures
All Authors: No reported disclosures
267. Small colony variants causing Prosthetic joint infection depict a different gene expression profile
Authors: Fernández-Rodríguez Diana , Carlos-Escalante José Alberto , Esaú López-Jácome Luis , Adriana Colin-Castro Claudia , Hernández-Durán Melissa , García-Contreras Rodolfo , Maeda Toshinari , Delgado Gabriela , Morales-Espinosa María del Rosario , Franco-Cendejas Rafael 
Published: November 2023

Abstract

Background
Prosthetic joint infection (PJI) is an infectious complication after total joint arthroplasty (TJA) with a high socioeconomic impact. Staphylococcus species attain for more than half of PJI cases. Biofilm formation and the development of small colony variants (SCV) have proven to enhance chronic and/or relapsing bacterial infections. In this regard, a better understanding of these factors can help overcome PJI and other device-associated infections. Thus, we aimed to analyze the clinical and microbiological differences between common/wild type (WT) and SCV Staphylococcus epidermidis strains

Methods
We analyzed a monomicrobial cohort of PJI patients affected by S. epidermidis. The bacterial isolates of patients with more than one-year of follow-up were examined for SCV detection. Then, we determined the genetic relatedness between strains with a pulsed field gel electrophoresis (PFGE). Finally, we selected 4 representative strains (2 WT and 2 SCV strains) to perform a differential expression analysis by RNA-seq. We performed a multiple test correction by controlling the false discovery rate (FDR) at 10%.

Results
S. epidermidis SCV affected 16 (37.6%) patients with monomicrobial PJI. The DNA fingerprints showed a high similarity, according to the Dice coefficient, between SCV strains and their WT counterparts. Nevertheless, further experiments demonstrated 22 genes with a significant differential expression profile: 12 were associated to metabolic pathways (carbon, amino acids, nucleotide, purines) and biosynthesis of secondary metabolites and cofactors, 3 with transmembrane transportation, 2 with redox balance, 1 with pH balance, 1 with integral components of the membrane and cell adhesion. Function was not identified for 3 of these genes.
Differential gene expression profile among SCV isolates causing prosthetic joint infection.

Conclusion
SCV are common in device-related infections like PJI. S. epidermidis SCV and their WT counterparts showed an extremely similar genetic background: still, SCV strains have a different gene expression profile. These findings explain some phenotypic variations (growth rate, biofilm properties, antimicrobial susceptibility profiles) found in clinical SCV strains. These genes may be promising therapeutic targets for combating chronic and/or relapsing bacterial infections.

Disclosures
All Authors: No reported disclosures
268. Human Mesenchymal Stem Cells Augment Neutrophil Function to the Human Fungal Pathogen, Candida albicans
Authors: Floyd Daniel , Tan Yuan , Souza-Moreira Luciana , Mei Shirley H J , Timmer Kyle , Mansour Michael 
Published: November 2023

Abstract

Background
Neutrophils are critical innate immune cells that play a crucial role in protection against invading pathogens. Mesenchymal stem cells (MSC) are multipotent progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes and possess immunomodulatory effects. MSC are the focus of clinical investigation in various comorbidities, including diabetes, cancer, and invasive infections.

Methods
Human MSC were derived from bone marrow and have been characterized to meet all criteria of MSC (ISCT guideline). Cells were seeded at 100,000 cells per well in fibronectin (50ug/mL)-coated 24-well plate and incubated for 24 hours at 37°C, 5% CO2. The next day, 0.5 mL of whole blood from three healthy donors was added for another 24 hours, after which the neutrophils were isolated, viability measured, and a high throughput multiparametric neutrophil function assay was performed, compared to non-MSC exposed neutrophils (control neutrophils). Functions tested included ROS (dihydrorhodamine 123 (DHR123), phagocytosis of iRFP-expressing Candida albicans, ectodomain shedding (loss of CD62L), and degranulation (increased expression of CD66b). Multiparametric assays were measured on a BD FACS Celesta 4 laser flow cytometer and data was analyzed on FlowJo software.

Results
Neutrophils co-cultured with MSC had an average viability of 96.3% compared to control neutrophils at 92.15%. Using the multiparametric neutrophil functional assay, neutrophils exposed to MSC ex vivo had increased functional responses to the fungal pathogen Candida albicans. MSC-conditioned neutrophils exhibited a 33.2% increase in degranulation, a 13.4% increase in ROS generation, and 16.7% additional phagocytosis compared to non-MSC neutrophil (control). Compared to fresh neutrophils isolated the day of testing, MSC-conditioned neutrophils had an average increase in phagocytosis, degranulation, shedding, and ROS generation of 36.9%, 62.7%, 50.3%, and 37.8%, respectively.

Conclusion
MSC increase the functional effector activity of neutrophils in healthy controls, specifically ROS generation, phagocytosis, ectodomain shedding, and degranulation in response to C. albicans. These data raise the possibility of MSC as a novel therapeutic intervention for patients with invasive infections.

Disclosures
Michael Mansour, MD, PhD, Thermofisher: Grant/Research Support
271. Epidemiology of ESBL Klebsiella pneumoniae Clonal Group 307 in Stony Brook, NY
Authors: Glicksberg Jessica , Sajib Monirul I , Sankaran Sanjana , Lindner Marissa , Mui Jonathan , Boniche-Alfaro Camila , Fries Bettina F 
Published: November 2023

Abstract

Background
According to the most recent CDC threat report infections caused by ESBL and carbapenem-resistant Klebsiella pneumoniae (CRKp) remain a persistent challenge. In the US dissemination of CRKp has been largely attributed to clonal group (CG) 258 with recent evidence that CG307, a dominant Klebsiella Extended Spectrum Beta-Lactamase (ESBL-Kp) clone is also emerging as CRKp. CG307 strains uniformly express a wzi-173 type capsular polysaccharide (CPS) whereas other Klebsiella strains including those of the CG258 express a wide variety of wzi-type CPS.
A paper from Houston, TX provided evidence of divergent evolution between CG258 and CG307. CG258 evolves upfront as carbapenem resistant whereas CG307 may initially emerge as ESBL. CR-Kp that belong to the CG307 have also been identified in SBU. Our objective was to determine if among ESBL-Kp CG307 is a common clone and to characterize the demographics of the infected patient population.

Methods
Molecular typing of the wzi gene alleles of 78 sequentially collected ESBL-Kp was performed. Demographic, microbiological and clinical data were extracted from the medical record.

Results
Wzi typing indicated that 43% of ESBLKp strains express a wzi173 type CPS indicating they belong to CG307 the remainder of ESBL strains expressed variable wzi-types. Preliminary data analysis showed that wzi173 expressing ESBL strains were predominantly derived from urine. The median age of patients was 64 years old with male predominance (60.8% males). The average length of stay is 41.6 days. Among these patients, 30.4% required ICU admission. Sepsis criteria were met in 56.5% of these patients within 2 days of positive culture. The mortality rate was 21.7%.

Conclusion
Our data provides evidence that CG307 is the most dominant ESBL Kp clone in hospitalized patients at Stony Brook University. Although the majority of cultures were from urinary source, ICU admission rates and mortality rates were also elevated. These data raise concern that high prevalence of ESBL CG307 in hospitalized patients may ultimately lead to emergence of carbapenem resistant CG307 strains especially if they emerge in patients who are treated with broad spectrum antibiotics.

Disclosures
All Authors: No reported disclosures
274. Potential HIV vaccine gp41 epitope targeting antibodies identify peptides with similarity to proposed Kawasaki disease related peptide, suggesting non-specific mimotope targeting of acidic amino acid enriched regions
Authors: Hakimuddin Sojar , Baron Sarah , Hicar Mark D 
Published: November 2023

Abstract

Background
We have previously isolated a highly mutated (83% homologous to predicted heavy chain germline) antibody (Ab) termed C group 76-Q13-6F5 (6F5) that targets a conformational epitope on gp41. 6F5, though non-neutralizing, has the capacity to mediate Ab dependent cell cytotoxicity (ADCC). When the variable chain (predicted to be VH1-02 derived) was mutated to germline (termed C group 76 ancestor, or 76Canc), surprisingly this Ab still exhibited significant ADCC activity. Many HIV vaccine strategies are focused on raising highly mutated Abs. We propose that there would be an advantage to developing vaccines related to epitopes that permit functional targeting by Abs using germline variable gene sequences.

Methods
To explore potential protein targets for vaccination strategies to raise and develop such Abs, we interrogated a peptide array of 29,127 linear peptides using PEPperCHIP® Human Epitome Microarray. We then confirmed peptide binding by Western blot and ELISAs. We also assessed binding to CDI laboratories HuProt protein microarray, containing &gt; 21,000 human proteins.

Results
76Canc specifically recognized a number of peptides enriched for glutamic and aspartic acid residues (top hit DEEEEYDEDEYEYDE). Meme analysis of positive peptides revealed a peptide sequence most similar to Hepatitis C virus, similar to a peptide implicated in Kawasaki disease (KD). We confirmed specific binding of four of the top peptide hits, including hepatitis C peptide recognition. We then confirmed binding of 76Canc-related Abs to a published optimized KD related peptide (KPAVIPDREALYQDIDEMEEC). Serum from KD and infectious controls was used to compete with biotinylated 76Canc-related Abs. Serum Abs targeting this epitope showed no specific correlation to having KD. Autoantigen screening of 76Canc identified a single human protein of interest that did contain acidic amino acid rich regions.Figure 1:HIV-1 gp41 antibodies recognize peptides similar to peptide implicated in Kawasaki Disease

Conclusion
This study reveals acidic motif targeting by specific anti-gp41 Abs and the derived germline Ab, but no evidence that these Abs are related to inflammation similar to KD. Cautious development of targeting such Abs by vaccination is warranted. Future structural comparison of these peptides with native proteins and binding competition studies are needed to confirm mimotope binding.

Disclosures
Mark D. Hicar, MD/PhD, Pfizer: site investigator for 2 trial
277. A Functional OMICs Evaluation of Omadacycline to Understand Anti-Clostridioides difficile Protective Effects
Authors: Jo Jinhee , Agyapong Samantha , Hu Chenlin , Wang Weiqun , Gonzales-Luna Anne J , Lancaster Chris , Garey Kevin W 
Published: November 2023

Abstract

Background
Omadacycline is an aminomethylcycline tetracycline with potent in vitro activity against C. difficile (CD) and a low propensity for CD infection (CDI) in clinical trials. In addition to potent in vitro activity, ideal properties of anti-CDI antibiotics include favorable effects on bile acids and short-chain fatty acids (SCFA). The aim of this study was to compare bile acid and SCFA changes in healthy volunteers given oral omadacycline versus vancomycin.

Methods
This was a phase 1 healthy volunteer study in 16 adults randomized to an oral 10-day course of either omadacycline or vancomycin. Stool samples were collected daily with metagenomics performed targeting the V4 region of the 16S ribosomal RNA gene using the Miseq platform (Illumina). Stool samples collected at baseline, during therapy, and follow-up visits were used for this functional omics evaluation. Targeted bile acids and SCFA quantification were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The final concentration of each gut metabolome was normalized by the corresponding stool sample weight.

Results
Both antibiotic groups showed a significant decrease in alpha diversity during therapy. Primary bile acids increased in vancomycin-treated subjects which was not observed in the omadacycline group although secondary bile acids diminished equally. Fecal SCFA concentrations of butyrate decreased in both groups while acetate and propionate were preserved in the omadacycline group compared to vancomycin.

Conclusion
Both omadacycline and vancomycin demonstrated changes in the gut microbiome with omadacycline showing favorable effects on bile acids and SCFA. These results provide mechanistic understandings on the anti-CDI properties of omadacycline.

Disclosures
Anne J. Gonzales-Luna, PharmD, BCIDP, Cidara Therapeutics: Grant/Research Support|Ferring Pharmaceuticals: Personal Fees|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support Kevin W. Garey, MS;PharmD, Paratek Pharmaceuticals: Grant/Research Support
281. Mathematical modeling of corticosteroid induced secondary infection and In silico Dose exploration of Dexamethasone, Hydrocortisone
Authors: Kim Dong-Hyun , Kyuong Chung Eun 
Published: November 2023

Abstract

Background
The use of steroids in infected patients is necessary to modulate the inflammatory response that is triggered by infection. However, there is a concern that steroid use could increase the risk of secondary infection. Here, To quantitatively and mechanistically evaluate the risk of secondary infection, we simulate different types and doses of steroids by utilizing a state-of-the-art hybrid PBPK-QSP modeling approach.

Methods
A minimal PBPK model of vancomycin and dexamethasone/hydrocortisone was developed and combined with a mathematical QSP model of bacterial infection and immune response. The models were linked to each other in R 4.2.2 using the rxode2 package. Usual dosing regimen of vancomycin and steroids were simulated, and long-term use of high-dose steroid therapy with vancomycin was also simulated. Vancomycin dosing was fixed at 1500mg IV infusion q12h for 7 days, dexamethasone 5 mg or 70 mg q12h IV infusion for 7 days, and hydrocortisone 100 mg or 500 mg q12h IV infusion for 7 days, with 1.2 times of the maintenance dose given as a loading dose. Plasma and lung PK profiles were derived using the minimal PBPK model to be combined seamlessly with the QSP model. Model-based simulations were conducted over a period of 1000 hours.

Results
Our minimal PBPK model simulated the PK profiles of vancomycin as well as dexamethasone and hydrocortisone in plasma and lung within the physiologically plausible range (Figure 1). In the case of dexamethasone 70 mg, the number of neutrophils decreased by about half compared to the case of dexamethasone 5 mg, and the number of bacteria in the lung compartment substantially increased (13.6% of the initial inoculum) after 300 hours (Figure 2). The other regimens did not show a significant increase in the number of bacteria.
Figure 1. The Plasma and PK profiles of Dexamethasone, Hydrocortisone and Vancomycin by using PBPK models. Figure 2. In silico simulation results of Bacterial infection and immune response during concomitant use of vancomycin and steroids.

Conclusion
Our modeling results suggest that the usual dose of steroid commonly used in infected patients does not increase the risk of secondary infection. However, long-term use of high-dose steroid therapy could increase the risk of secondary infection. When long-term use of high-dose steroid therapy is needed, Hydrocortisone could be a better option than dexamethasone from the perspective of infection risk

Disclosures
All Authors: No reported disclosures
283. Screening the PE/PPE secreted effectors of Mycobacterium tuberculosis to uncover novel virulence-promoting genes
Authors: Koleske Benjamin , Shen Jessica , Gupta Manish , Bishai William 
Published: November 2023

Abstract

Background
Mycobacterium tuberculosis (M.tb) remains the leading cause of infectious disease mortality, with over 10 million active cases and 1.5 million deaths annually at the global scale. Although M.tb infection prompts highly inflammatory granulomatous lesions in the lung, the bacterium is not eradicated by the immune cell infiltrate. Instead, M.tb uses numerous virulence factors to facilitate growth within macrophages, including secretory proteins that subvert host immune processes. In this work, we examined the substrates of ESX-5, a prolific type VII secretion system unique to slow-growing pathogenic mycobacteria, for their roles in contributing to bacterial virulence in a mouse lung infection model. These secretory substrates, the PE and PPE family proteins (named for conserved Pro-Glu and Pro-Pro-Glu domains), have evolutionarily expanded from 11 members in the harmless commensal M. smegmatis to 167 members in the M.tb H37Rv reference strain, suggesting they serve critical functions for an intracellular pathogen with an otherwise reduced genome. Certain PE/PPE genes have been implicated in a broad range of immune relevant functions, including TLR signaling, apoptosis inhibition, and NF-κB activation, though the vast majority remain understudied.
M.tb secretion during infection
(A) Following phagocytosis by a macrophage, M.tb blocks phagosome maturation and permeabilizes the phagosome to secrete effectors into the macrophage cytoplasm. (B) The ESX-5 system secretes a large (&gt; 150) repertoire of PE/PPE proteins predicted to alter host signaling pathways.

Methods
We performed Tn-Seq in the TB mouse model using a pooled library of 81 M.tb strains with single transposon insertions in known PE/PPE genes, all of which show no in vitro growth defect, to identify mutants that demonstrate an in vivo fitness cost.

Results
Bacterial genomes were harvested from the lungs of aerosol-infected BALB/c mice at acute (1 week) and subacute (3 week) timepoints and compared to the composition of the initial pool (day 1). Deep sequencing demonstrated significant decreases in the prevalence of 20 strains over time; furthermore, the findings were highly reproducible across all animals (n=10 per timepoint), showed time-dependent kinetics, and identified several PE/PPE genes previously found to contribute to M.tb pathogenesis.

Conclusion
Our mouse Tn-Seq screen of 81 M.tb PE/PPE mutants identified 20 mutants that were attenuated for growth in mice in a pooled competition format. Thus, a high proportion of PE/PPE genes may be virulence-promoting factors.

Disclosures
All Authors: No reported disclosures