A Neonatal Model of Intravenous Staphylococcus epidermidis Infection in Mice <24 h Old Enables Characterization of Early Innate Immune Responses

Staphylococcus epidermidis (SE) causes late onset sepsis and significant morbidity in catheterized preterm newborns. Animal models of SE infection are useful in characterizing disease mechanisms and are an important approach to developing improved diagnostics and therapeutics. Current murine models of neonatal bacterial infection employ intraperitoneal or subcutaneous routes at several days of age, and may, therefore, not accurately reflect distinct features of innate immune responses to bacteremia. In this study we developed, validated, and characterized a murine model of intravenous (IV) infection in neonatal mice <24 hours (h) old to describe the early innate immune response to SE. C57BL/6 mice <24 h old were injected IV with 106, 107, 108 colony-forming units (CFU) of SE 1457, a clinical isolate from a central catheter infection. A prospective injection scoring system was developed and validated, with only high quality injections analyzed. Newborn mice were euthanized between 2 and 48 h post-injection and spleen, liver, and blood collected to assess bacterial viability, gene expression, and cytokine production. High quality IV injections demonstrated inoculum-dependent infection of spleen, liver and blood. Within 2 h of injection, SE induced selective transcription of TLR2 and MyD88 in the liver, and increased systemic production of plasma IL-6 and TNF-α. Despite clearance of bacteremia and solid organ infection within 48 h, inoculum-dependent impairment in weight gain was noted. We conclude that a model of IV SE infection in neonatal mice <24 h old is feasible, demonstrating inoculum-dependent infection of solid organs and a pattern of bacteremia, rapid and selective innate immune activation, and impairment of weight gain typical of infected human neonates. This novel model can now be used to characterize immune ontogeny, evaluate infection biomarkers, and assess preventative and therapeutic modalities

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10&nbsp;years; 78.2% included were male with a median age of 37&nbsp;years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Morphine inhibition of macrophage phagocytosis and bactericidal functions.

University of Minnesota Ph.D. dissertation. January 2011. Major: Pharmacology. Advisor: Sabita Roy, PhD. 1 computer file (PDF); xiii, 158 pages.For centuries, opioids have been implicated in increasing susceptibility to infection, reducing bacterial clearance, and increasing bacterial dissemination. Macrophages as key cells of innate immunity play an essential role in pathogen clearance and antigen presentation. Macrophage phagocytosis is a key mechanism responsible for host defense against bacterial pathogens. Although it is known that opioid addicts are prone to both bacterial and viral infections, the molecular and cellular mechanisms underlying these processes remain to be elucidated. Therefore the goal of this research was to investigate mechanisms of decreased bacterial clearance as a contributing factor in the increased susceptibility to infection in opiate drug abusers. To this end, first set of studies examined the role of morphine on inhibition of key mechanisms involved in Fc-gamma receptor mediated phagocytosis. It was demonstrated that morphine inhibits phagocytosis by inhibiting actin polymerization through a cAMP, PKA and MAPK dependant pathways. By superactivation of adenylyl cyclase morphine increases intracellular cAMP leading to inhibition of actin polymerization. Furthermore, morphine by inhibiting p38 MAPK and ERK 1/2 MAPK causes inhibition of actin polymerization and phagocytosis. By modulating TLR4 receptor function morphine was also able to increase macrophage phagocytosis, indicating that morphine might have a differential effect on internalization of Gram-positive, versus Gram-negative pathogens. These effects were mediated through a MyD88 and p38 MAPK dependant pathways leading to changes in actin polymerization and phagocytosis. In addition to macrophage's ability to internalize pathogens, elimination of internalized pathogen is essential for effective bacterial clearance. We therefore set out to investigate morphine's modulation of macrophage bactericidal mechanisms. We note that morphine inhibits bacterial killing by inhibiting essential mechanisms involved in this process such as formation of reactive oxygen intermediates, reactive nitrogen intermediates, as well as phago-lysosomal fusion. Morphine by inhibiting these essential mechanisms impedes eradication of bacterial infections and leads to detrimental consequences for the host. These series of studies have extended our knowledge in an underrepresented yet clinically significant field of study, however many questions still remain to be addressed and it is crucial to investigate the answers given the prevalence of morphine use today

Morphine withdrawal inhibits IL-12 induction in a macrophage cell line through a mechanism that involves cAMP

There are very few studies that examine the effects that morphine withdrawal has on immune functioning, and of these even fewer describe the mechanisms by which withdrawal brings about these changes. Our previous work demonstrated that morphine withdrawal contributed to Th cell differentiation by biasing cells toward the Th2 lineage. A major finding from these studies was that IL-12 was decreased following withdrawal, and it was concluded that this decrease may be a mechanism by which morphine withdrawal is mediating Th2 polarization. Therefore, it was the aim of the current studies to develop an in vitro model to examine the process of morphine withdrawal and to understand the signaling mechanisms that withdrawal may use to effect IL-12 production through the use of this model. It was demonstrated and concluded that morphine withdrawal may be effecting IL-12 production by increasing cAMP levels, which activates protein kinase A. Protein kinase A activation then prevents the phosphorylation and subsequent degradation of IkappaB, which in turn prevents translocation of the NF-kappaB p65 subunit to the nucleus to transactivate the IL-12 p40 gene, ultimately resulting in decreased IL-12 production following LPS stimulation

Differential effects of gram-positive and gram-negative bacterial products on morphine induced inhibition of phagocytosis

Opioid drug abusers have a greater susceptibility to gram positive (Gram (+)) bacterial infections. However, the mechanism underlying opioid modulation of Gram (+) versus Gram (−) bacterial clearance has not been investigated. In this study, we show that opioid treatment resulted in reduced phagocytosis of Gram (+), when compared to Gram (−) bacteria. We further established that LPS priming of chronic morphine treated macrophages leads to potentiated phagocytosis and killing of both Gram (+) and Gram (−) bacteria in a P-38 MAP kinase dependent signaling pathway. In contrast, LTA priming lead to inhibition of both phagocytosis and bacterial killing. This study demonstrates for the first time the differential effects of TLR4 and TLR2 agonists on morphine induced inhibition of phagocytosis. Our results suggest that the incidence and severity of secondary infections with Gram (+) bacteria would be higher in opioid abusers

SE inoculum-dependent transcription of TLR signaling pathway genes.

<p>Neonatal mice were injected IV with SE at A) 10⁶, B) 10⁷, and C) 10⁸ CFU and euthanized at 2 h. Liver tissue was harvested, RNA isolated and transcription of TLR-signaling pathway genes assessed. Log₁₀ normalized gene expression levels are shown for both saline (x-axis) and inoculum SE (y-axis) injected mice. Inoculum-dependent up-regulation of TLR-gene transcription is evident, with mRNA transcripts that are significantly up- or down-regulated shown in red.</p

Induction of bacteremia is proportional to injection scoring criteria.

<p>Newborn mice less than 24 h old were injected with 10⁸ CFU of SE. Mice were euthanized, organs harvested, and CFUs measured. CFU data were stratified by injection score, determined as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043897#pone-0043897-t002" target="_blank">Table 2</a>. Prospective injection scores of 3 or higher correlated with substantially greater CFUs in mouse spleen and liver (N = 5–8, ** p<0.01, *** p<0.001, Mann-Whitney t-test).</p

Injection scoring criteria.

<p>Injection scoring criteria.</p