Clostridium difficile Infection and Patient-Specific Antimicrobial Resistance Testing Reveals a High Metronidazole Resistance Rate.

BACKGROUND: Clostridium difficile (CD) infection (CDI) causes marked morbidity and mortality, accounting for large healthcare expenditures annually. Current CDI treatment guidelines focus on clinical markers of patient severity to determine the preferred antibiotic regimen of metronidazole versus vancomycin. The antimicrobial resistance patterns for patients with CD are currently unknown. AIM: The aim of this study was to define the antimicrobial resistance patterns for CD. METHODS: This study included all patients with stools sent for CD testing to a private laboratory (DRG Laboratory, Alpharetta, Georgia) in a 6-month period from across the USA. Patient data was de-identified, with only age, gender, and zip-code available per laboratory protocol. All samples underwent PCR testing followed by hybridization for CD toxin regions A and B. Only patients with CD-positive PCR were analyzed. Antimicrobial resistance testing using stool genomic DNA evaluated presence of imidazole- and vancomycin-resistant genes using multiplex PCR gene detection. RESULTS: Of 2743, 288 (10.5%) stool samples were positive for CD. Six were excluded per protocol. Of 282, 193 (69.4%) were women, and average age was 49.4 ± 18.7 years. Of 282, 62 were PCR positive for toxins A and B, 160 for toxin A positive alone, and 60 for toxin B positive alone. Antimicrobial resistance testing revealed 134/282 (47.5%) patients resistant to imidazole, 17 (6.1%) resistant to vancomycin, and 9 (3.2%) resistant to imidazole and vancomycin. CONCLUSIONS: CD-positive patients with presence of imidazole-resistant genes from stool DNA extract was a common phenomenon, while vancomycin resistance was uncommon. Similar to treatment of other infections, antimicrobial resistance testing should play a role in CDI clinical decision-making algorithms to enable more expedited and cost-effective delivery of patient care.

Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition.

Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, α-smooth muscle actin (α-SMA), and vimentin by over 200% (P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ∼50% and α-SMA was increased by 100% (P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (25-50 µM) activated endogenous transforming growth factor-ß1 (TGF-ß1), as evident by H2DCFDA immunofluorescence and ELISA (P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-ß1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated α-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-ß1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ∼10-fold (P < 0.05) and that this effect was prevented by intraperitoneal TGF-ß1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-ß1 signaling.

Safety monitoring of bivalent mRNA COVID-19 vaccine among pregnant persons in the vaccine adverse event reporting System - United States, September 1, 2022 - March 31, 2023.

BACKGROUND: Pregnant persons are at increased risk of severe COVID-19 illness. Bivalent mRNA COVID-19 vaccination is recommended for everyone, including pregnant persons. However, data are limited on the safety of bivalent mRNA COVID-19 vaccination during pregnancy. OBJECTIVE: To evaluate and summarize reports to the Vaccine Adverse Event Reporting System (VAERS), a national spontaneous reporting system, among pregnant persons who received bivalent mRNA COVID-19 vaccine. METHODS: VAERS U.S. reports of adverse events (AEs) in pregnant persons who received the bivalent mRNA COVID-19 vaccine from 9/1/2022-03/31/2023 were identified. Clinicians reviewed all reports and available medical records. AEs of these reports were compared with AEs reported to VAERS following monovalent mRNA COVID-19 booster vaccination in pregnancy. RESULTS: VAERS received 136 reports for pregnant persons who received bivalent mRNA COVID-19 vaccine; 87 (64 %) after BNT162b2 (Pfizer-BioNTech), and 48 (35 %) after mRNA-1273 (Moderna); 28 (20.6 %) reports were classified as serious. The most common pregnancy-specific outcomes reported included 12 (8.8 %) spontaneous abortions (<20 weeks gestation), 6 (4.4 %) episodes of preterm delivery, and 5 (3.7 %) reports of preeclampsia. One stillbirth (≥20 weeks gestation) was reported. No maternal or infant deaths were reported. There were 6 reports of AEs in infants, which included 3 reports of admissions to the neonatal intensive care unit: two infants with low birth weight, and one infant with a patent ductus arteriosus and patent foramen ovale. Non-pregnancy-specific adverse events were mostly COVID-19 infection and systemic reactions (e.g., headache, fatigue). Pregnancy-specific conditions were reported less frequently after bivalent mRNA COVID-19 vaccination compared to monovalent mRNA COVID-19 booster vaccination (3rd and 4th dose). CONCLUSIONS: Based on this review of reports to VAERS, the safety profile of bivalent mRNA COVID-19 vaccination in pregnant persons was comparable to that observed for monovalent mRNA COVID-19 booster vaccination (3rd and 4th dose) in pregnant persons.

The glucagon-like peptide-1 receptor agonist Exendin 4 has a protective role in ischemic injury of lean and steatotic liver by inhibiting cell death and stimulating lipolysis.

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase - all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.

TLR5 or NLRC4 is necessary and sufficient for promotion of humoral immunity by flagellin.

The fact that some TLR-based vaccine adjuvants maintain function in TLR-deficient hosts highlights that their mechanism of function remains incompletely understood. Thus, we examined the ability of flagellin to induce cytokines and elicit/promote murine antibody responses upon deletion of the flagellin receptors TLR5 and/or NLRC4 (also referred to as IPAF) using a prime/boost regimen. In TLR5-KO mice, flagellin failed to induce NF-κB-regulated cytokines such as keratinocyte-derived chemokine (CXCL1) but induced WT levels of the inflammasome cytokine IL-18 (IL-1F4). Conversely, in NLRC4-KO mice, flagellin induced keratinocyte-derived chemokine, but not IL-18, whereas TLR5/NLRC4-DKO lacked induction of all cytokines measured. Flagellin/ovalbumin treatment resulted in high-antibody titers to both flagellin and ovalbumin in WT, TLR5-KO and DKO mice but did not elicit antibodies to either in TLR5/NLRC4-DKO mice. Thus, flagellin's ability to elicit/promote humoral immunity requires a germ-line-encoded receptor capable of recognizing this molecule. Such promotion of adaptive immunity can be effectively driven by either TLR5-mediated activation of NF-κB or NLRC4-mediated activation of the inflammasome.

Interaction between activated chemokine receptor 1 and FcepsilonRI at membrane rafts promotes communication and F-actin-rich cytoneme extensions between mast cells.

Chemokines play important regulatory roles in immunity, but their contributions to mast cell function remain poorly understood. We examined the effects of FcepsilonRI-chemokine receptor (CCR) 1 co-stimulation on receptor localization and cellular morphology of bone marrow-derived mast cells. Whereas FcepsilonRI and CCR1 co-localized at the plasma membrane in unsensitized cells, sensitization with IgE promoted internalization of CCR1 molecules. Co-stimulation of FcepsilonRI and CCR1 with antigen and macrophage inflammatory protein-1alpha was more effective than FcepsilonRI stimulation alone in causing leading edge formation, flattened morphology, membrane ruffles and ganglioside (GM1(+)) lipid mediator release. Co-stimulation resulted in phalloidin-positive cytoneme-like cellular extensions, also known as tunneling nanotubes, which originated at points of calcium accumulation. This is the first report of cytoneme formation by mast cells. To determine the importance of lipid rafts for mast cell function, the cells were cholesterol depleted. Cholesterol depletion enhanced degranulation in resting, sensitized and co-stimulated cells, but not in FcepsilonRI-cross-linked cells, and inhibited formation of filamentous actin(+) cytonemes but not GM1(+) cytonemes. Treatment with latrunculin A to sequester globular-actin abolished cytoneme formation. The cytonemes may participate in intercellular communication during allergic and inflammatory responses, and their presence in the co-stimulated mast cells suggests new roles for CCRs in immunopathology.

CCR1 expression and signal transduction by murine BMMC results in secretion of TNF-alpha, TGFbeta-1 and IL-6.

Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with various diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine IL-3 expressed CCR1 after 5-6 weeks. We also report for the first time that mBMMC(CCR1+) cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1alpha and the IgE receptor FcepsilonRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcepsilonRI stimulation alone, as assessed by beta-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca(2+) influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1-FcepsilonRI co-stimulation. These factors include transforming growth factor beta-1, tumor necrosis factor-alpha and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.

Functional regulation of human neutrophil Fc gamma receptors.

Interaction between Fc receptors expressed on phagocytic cells and antibodies play a critical role in innate immune response. Interestingly, immune cells such as neutrophils, monocytes, and dendritic cells (DCs) express multiple Fc receptors for IgG (FcgammaR) with overlapping ligand specificity. These receptors compete for the same ligand on the target and are known to transduce positive and negative signals to the same cell, depending on presence of type of signaling motif in their cytoplasmic domain. Neutrophils, the first line of defense against bacterial infection and the major phagocytic cell in the blood, express two types of FcgammaRs depending on the species. In humans, the neutrophils co-express immunoreceptor tyrosine-based activation motif (ITAM) containing CD32A and glycosyl-phosphatidyl inositol (GPI)-anchored CD16B, which is in contrast to co-expression of ITAM containing CD16A and ITIM containing CD32B in mouse neutrophils. Recent studies in gene knockout mice have demonstrated that the negative signaling by CD32B plays a critical role in preventing immune complex (IC)-mediated autoimmune diseases by regulating the activation signal delivered by CD16A. However, it is not known how the function of ITAM signaling CD32A is regulated in human neutrophils. Recent observations from our laboratory suggest that in human neutrophils, the CD32A receptor is regulated at the ligand-binding stage. Using a CD16B-deficient donor, we found that the CD32A expressed on resting neutrophils is unable to bind ligand; however, once neutrophils are activated with fMLP, a bacterial chemotactic peptide, the CD32A is functionally active in binding ligand. We also observed that this regulation is neutrophil-specific phenomenon. These observations suggest that FcgammaR can be regulated by distinct mechanisms and factors such as membrane-anchoring, cell-specific signaling, and avidity modulation that may be coordinately involved in regulating the function of human FcgammaR. Because neutrophils may be activated during infectious and inflammatory diseases, the knowledge of functional regulation of FcgammaR will be useful in designing therapies for many autoimmune diseases.

Hemolytic and nonhemolytic vancomycin-resistant Enterococcus faecalis isolated from beef imported to Malaysia.

Twenty-two strains of vancomycin-resistant Enterococcus faecalis were isolated from 9 (6%) of 150 samples of frozen beef and beef products imported to Malaysia. The isolates were obtained from eight samples of beef and one sample of minced beef patty. No E. faecalis was isolated from frankfurters. Twelve of the 22 isolates (54.5%) were beta-hemolytic, and all isolates harbored the vanA gene. All vancomycin-resistant isolates were also resistant to streptomycin, erythromycin, kanamycin, bacitracin, ceftazimide, gentamycin, tetracycline, nalidixic acid, and teicoplanin; 95.4% were resistant to trimethoprimsulfamethoxazole; 68.8% were resistant to chloramphenicol; and 41% were resistant to ampicillin and penicillin. Small plasmids ranging in size from 1.5 to 5.8 kb were detected in 8 (36.4%) of 22 strains. The 22 isolates were classified into 20 random amplified polymorphic DNA types. Isolates were divided into two groups, each containing subclusters, that may reflect their clonal lineages. It is concluded that several clones of vancomycin-resistant E. faecalis are represented in the isolates obtained from beef imported to Malaysia.

Rotavirus-induced IFN-ß promotes anti-viral signaling and apoptosis that modulate viral replication in intestinal epithelial cells.

Rotavirus (RV), a leading cause of diarrhea, primarily infects intestinal epithelial cells (IEC). Rotavirus-infected IEC produce IFN-ß and express hundreds of IFN-dependent genes. We thus hypothesized that type 1 IFN plays a key role in helping IEC limit RV replication and/or protect against cell death. To test this hypothesis, we examined IEC (HT29 cells) infected with RV (MOI 1) ± neutralizing antibodies to IFN-α/ß via microscopy and SDS-PAGE immunoblotting. We hypothesized that neutralization of IFN would be clearly detrimental to RV-infected IEC. Rather, we observed that blockade of IFN function rescued IEC from the apoptotic cell death that otherwise would have occurred 24-48 h following exposure to RV. This resistance to cell death correlated with reduced levels of viral replication at early time points (< 8 h) following infection and eventuated in reduced production of virions. The reduction in RV replication that resulted from IFN neutralization correlated with, and could be recapitulated by, blockade of IFN-induced protein kinase R (PKR) activation, suggesting involvement of this kinase. Interestingly, pharmacologic blockade of caspase activity ablated RV-induced apoptosis and dramatically increased viral protein synthesis, suggesting that IFN-induced apoptosis helps to control RV infection. These results suggest non-mutually exclusive possibilities that IFN signaling is usurped by RV to promote early replication and induction of cell death may be a means by which IFN signaling possibly clears RV from the intestine.

Signal-specific activation and regulation of human neutrophil Fc gamma receptors.

FcgammaRs with the ITIM domain have been shown to regulate the inflammatory signal delivered by the ITAM-containing FcgammaRs. In this study, we demonstrate that the function of human neutrophil FcgammaR type IIA (CD32A) is regulated in a distinct manner by different cell activation signals at the ligand-binding stage. Activation of neutrophils with fMLP up-regulated the ligand-binding function of CD32A, whereas PMA-mediated activation completely abolished ligand binding without altering CD32A expression. Furthermore, PMA treatment also abolished CD16B-dependent ligand binding irrespective of the level of expression. The effect of PMA was cell type specific, because the ligand-binding function of CD32A expressed on cultured cells such as K562 and CHO-CD32A transfectants was not affected by PMA. Interestingly, phorbol 12,13-dibutyrate, another phorbol ester, and IL-8 up-regulated CD32A-dependent ligand-binding function. These results demonstrate that regulation of CD32A-dependent ligand binding in human neutrophils is not only cell type specific but also activation signal specific. Moreover, these results suggest the possibility that signals delivered to neutrophils by various inflammatory stimuli can exert opposing effects on the function of human FcgammaRs, representing a novel inside-out regulatory mechanism of FcgammaR ligand binding.