An in silico modeling approach to understanding the dynamics of the post-burn immune response.

Introduction: Burns are characterized by a massive and prolonged acute inflammation, which persists for up to months after the initial trauma. Due to the complexity of the inflammatory process, Predicting the dynamics of wound healing process can be challenging for burn injuries. The aim of this study was to develop simulation models for the post-burn immune response based on (pre)clinical data. Methods: The simulation domain was separated into blood and tissue compartments. Each of these compartments contained solutes and cell agents. Solutes comprise pro-inflammatory cytokines, anti-inflammatory cytokines and inflammation triggering factors. The solutes diffuse around the domain based on their concentration profiles. The cells include mast cells, neutrophils, and macrophages, and were modeled as independent agents. The cells are motile and exhibit chemotaxis based on concentrations gradients of the solutes. In addition, the cells secrete various solutes that in turn alter the dynamics and responses of the burn wound system. Results: We developed an Glazier-Graner-Hogeweg method-based model (GGH) to capture the complexities associated with the dynamics of inflammation after burn injuries, including changes in cell counts and cytokine levels. Through simulations from day 0 - 4 post-burn, we successfully identified key factors influencing the acute inflammatory response, i.e., the initial number of endothelial cells, the chemotaxis threshold, and the level of chemoattractants. Conclusion: Our findings highlight the pivotal role of the initial endothelial cell count as a key parameter for intensity of inflammation and progression of acute inflammation, 0 - 4 days post-burn.

Kinetics of Inflammatory Mediators in the Immune Response to Burn Injury: Systematic Review and Meta-Analysis of Animal Studies.

Burns are often accompanied by a dysfunctional immune response, which can lead to systemic inflammation, shock, and excessive scarring. The objective of this study was to provide insight into inflammatory pathways associated with burn-related complications. Because detailed information on the various inflammatory mediators is scattered over individual studies, we systematically reviewed animal experimental data for all reported inflammatory mediators. Meta-analyses of 352 studies revealed a strong increase in cytokines, chemokines, and growth factors, particularly 19 mediators in blood and 12 in burn tissue. Temporal kinetics showed long-lasting surges of proinflammatory cytokines in blood and burn tissue. Significant time-dependent effects were seen for IL-1ß, IL-6, TGF-ß1, and CCL2. The response of anti-inflammatory mediators was limited. Burn technique had a profound impact on systemic response levels. Large burn size and scalds further increased systemic, but not local inflammation. Animal characteristics greatly affected inflammation, for example, IL-1ß, IL-6, and TNF-α levels were highest in young, male rats. Time-dependent effects and dissimilarities in response demonstrate the importance of appropriate study design. Collectively, this review presents a general overview of the burn-induced immune response exposing inflammatory pathways that could be targeted through immunotherapy for burn patients and provides guidance for experimental set-ups to advance burn research.

Monocytes and T cells incorporated in full skin equivalents to study innate or adaptive immune reactions after burn injury.

Introduction: Thermal injury often leads to prolonged and excessive inflammation, which hinders the recovery of patients. There is a notable absence of suitable animal-free models for investigating the inflammatory processes following burn injuries, thereby impeding the development of more effective therapies to improve burn wound healing in patients. Methods: In this study, we established a human full skin equivalent (FSE) burn wound model and incorporated human peripheral blood-derived monocytes and T cells. Results: Upon infiltration into the FSEs, the monocytes differentiated into macrophages within a span of 7 days. Burn-injured FSEs exhibited macrophages with increased expression of HLA-DR+ and elevated production of IL-8 (CXCL8), in comparison to uninjured FSEs. Among the T cells that actively migrated into the FSEs, the majority were CD4+ and CD25+. These T cells demonstrated augmented expression of markers associated with regulatory T cell, Th1, or Th17 activity, which coincided with significant heightened cytokine production, including IFN-γ, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), and TGF-ß1. Burn injury did not impact the studied effector T cell subsets or cytokine levels. Discussion: Collectively, this study represents a significant advancement in the development of an immunocompetent human skin model, specifically tailored for investigating burn-induced innate or adaptive immune reactions at the site of burn injury.

The health and budget impact of sodium-glucose co-transporter-2 inhibitors (SGLT2is) in The Netherlands.

OBJECTIVES: Type-2 Diabetes mellitus (T2DM) increases both the patient risk of cardiovascular disease (CVD) and renal outcomes, such as chronic kidney disease (CKD). Recent clinical trials of the glucose-lowering drug-class of sodium-glucose co-transporter-2 inhibitors (SGLT2is) have shown benefits in preventing CVD events and progression of CKD, leading to an update of the Dutch T2DM treatment guideline for patients at risk. The aim of this study is to assess the health and economic impact of the guideline-recommended utilization of SGLT2is in the Netherlands. METHODS: The patient population at risk was determined by multiplying Dutch T2DM prevalence rates with the total numbers of inhabitants of the Netherlands in 2020. Subsequently, two analyses, comparing a treatment setting before and after implementation of the new guideline for SGLT2is, were conducted. Clinical and adverse event rates in both settings as well as direct healthcare costs were sourced from the literature. Total costs were calculated by multiplying disease prevalence, event rates and costs associated to outcomes. One-time disutilities per event were included to estimate the health impact. The potential health and economic impact of implementing the updated guideline was calculated. RESULTS: Using a 5-year time horizon, the guideline-suggested utilization of SGLT2is resulted in a health impact equal to 4835 quality adjusted life years gained (0.0031 per patient per year) and €461 million cost-savings. The costs of treatment with SGLT2is were €813 million. Hence the net budget impact was €352 million for the total Dutch T2DM population, which translated to €0,57 per patient per day. CONCLUSION: SGLT2is offer an option to reduce the number of CVD and CKD related events and associated healthcare costs and health losses in the Netherlands. Further research is needed to include the benefits of improved T2DM management options from a broader societal perspective.HighlightsThe glucose-lowering drug-class of sodium-glucose co-transporter-2 inhibitors (SGLT2is) has shown benefits in preventing cardiovascular events and progression of kidney disease in patients with type-2 diabetes leading to a revision of the respective Dutch treatment guideline.The 5-year budget impact of the adoption of SGLT2is in the new treatment guideline was equal to €352 million or €0.57 per patient per day, with a total of 4385 quality adjusted life years gained.The introduction of SGLT2is for Dutch type-2 diabetes patients has the potential to substantially reduce the number of cardiovascular as well as renal disease events and related healthcare costs while also delivering a health benefit.

Full Skin Equivalent Models for Simulation of Burn Wound Healing, Exploring Skin Regeneration and Cytokine Response.

Healing of burn injury is a complex process that often leads to the development of functional and aesthetic complications. To study skin regeneration in more detail, organotypic skin models, such as full skin equivalents (FSEs) generated from dermal matrices, can be used. Here, FSEs were generated using de-epidermalized dermis (DED) and collagen matrices MatriDerm® and Mucomaix®. Our aim was to validate the MatriDerm- and Mucomaix-based FSEs for the use as in vitro models of wound healing. Therefore, we first characterized the FSEs in terms of skin development and cell proliferation. Proper dermal and epidermal morphogenesis was established in all FSEs and was comparable to ex vivo human skin models. Extension of culture time improved the organization of the epidermal layers and the basement membrane in MatriDerm-based FSE but resulted in rapid degradation of the Mucomaix-based FSE. After applying a standardized burn injury to the models, re-epithelization occurred in the DED- and MatriDerm-based FSEs at 2 weeks after injury, similar to ex vivo human skin. High levels of pro-inflammatory cytokines were present in the culture media of all models, but no significant differences were observed between models. We anticipate that these animal-free in vitro models can facilitate research on skin regeneration and can be used to test therapeutic interventions in a preclinical setting to improve wound healing.

Burn-injured skin is marked by a prolonged local acute inflammatory response of innate immune cells and pro-inflammatory cytokines.

The systemic and local immune response in burn patients is often extreme and derailed. As excessive inflammation can damage healthy tissues and slow down the healing process, modulation of inflammatory responses could limit complications and improve recovery. Due to its complexity, more detailed information on the immune effects of thermal injury is needed to improve patient outcomes. We therefore characterized and quantified subsets of immune cells and mediators present in human burn wound tissue (eschar), sampled at various time points. This study shows that after burn injury, the number of immune cells were persistently increased, unlike the normal wound healing process. There was an immediate, strong increase in neutrophils and a moderate increase in monocytes/macrophages and lymphocytes, especially in the second and third week post burn. The percentage of classical (CD14highCD16-) monocytes/macrophages demonstrated a steady decrease over time, whereas the proportion of intermediate (CD14highCD16+) monocytes/macrophages slowly increased. The absolute numbers of T cells, NK cells and B cells increased up to week 3, while the fraction of γδ T cells was increased only in week 1. Secretome profiling revealed high levels of chemokines and an overall pro-inflammatory cytokine milieu in burn tissue. The local burn immune response shows similarities to the systemic immune reaction, but differs in neutrophil maturity and lymphocyte composition. Altogether, the neutrophil surges, high levels of pro-inflammatory cytokines and limited immunosuppression might be key factors that prolong the inflammation phase and delay the wound healing process in burns.

Burn-Induced Local and Systemic Immune Response: Systematic Review and Meta-Analysis of Animal Studies.

Because burn injuries are often followed by a derailed immune response and excessive inflammation, a thorough understanding of the occurring reactions is key to preventing secondary complications. This systematic review, which includes 247 animal studies, shows the postburn response of 14 different immune cell types involved in immediate and long-term effects in both wound tissue and circulation. Peripheral blood neutrophil and monocyte numbers increased directly after burns, whereas thrombocyte numbers increased near the end of the first week. However, lymphocyte numbers were decreased for at least 2 weeks. In burn wound tissue, neutrophil and macrophage numbers accumulated during the first 3 weeks. Burns also altered cellular functions because we found an increased migratory potential of leukocytes, impaired antibacterial activity of neutrophils, and enhanced inflammatory mediator production by macrophages. Neutrophil surges were positively associated with burn size and were highest in rats. Altogether, this comprehensive overview of the temporal immune cell dynamics shows that unlike normal wound healing, burn injury induces a long-lasting inflammatory response. It provides a fundamental research basis to improve experimental set-ups, burn care, and outcomes.

Selective tumor antigen vaccine delivery to human CD169+ antigen-presenting cells using ganglioside-liposomes.

Priming of CD8+ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+ CD169+ monocytes and Axl+ CD169+ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+ moDCs and Axl+ CD169+ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+ T cells. Finally, Axl+ CD169+ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+ DCs to drive antitumor T cell responses.

Persistent Systemic Inflammation in Patients With Severe Burn Injury Is Accompanied by Influx of Immature Neutrophils and Shifts in T Cell Subsets and Cytokine Profiles.

Severe burn injury causes local and systemic immune responses that can persist up to months, and can lead to systemic inflammatory response syndrome, organ damage and long-term sequalae such as hypertrophic scarring. To prevent these pathological conditions, a better understanding of the underlying mechanisms is essential. In this longitudinal study, we analyzed the temporal peripheral blood immune profile of 20 burn wound patients admitted to the intensive care by flow cytometry and secretome profiling, and compared this to data from 20 healthy subjects. The patient cohort showed signs of systemic inflammation and persistently high levels of pro-inflammatory soluble mediators, such as IL-6, IL-8, MCP-1, MIP-1ß, and MIP-3α, were measured. Using both unsupervised and supervised flow cytometry techniques, we observed a continuous release of neutrophils and monocytes into the blood for at least 39 days. Increased numbers of immature neutrophils were present in peripheral blood in the first three weeks after injury (0.1-2.8 × 106/ml after burn vs. 5 × 103/ml in healthy controls). Total lymphocyte numbers did not increase, but numbers of effector T cells as well as regulatory T cells were increased from the second week onward. Within the CD4+ T cell population, elevated numbers of CCR4+CCR6- and CCR4+CCR6+ cells were found. Altogether, these data reveal that severe burn injury induced a persistent innate inflammatory response, including a release of immature neutrophils, and shifts in the T cell composition toward an overall more pro-inflammatory phenotype, thereby continuing systemic inflammation and increasing the risk of secondary complications.

Synergistic microbicidal effect of cationic antimicrobial peptides and teicoplanin against planktonic and biofilm-encased Staphylococcus aureus.

Antibiotic resistance and biofilm formation are the main reasons for failure in treatment of bacterial infections. This study aimed to identify synergistic combinations of conventional antibiotics and novel synthetic antimicrobial and antibiofilm peptides (SAAPs) inspired by the structures of the natural human cationic peptides LL-37 and thrombocidin-1 (TC-1). The LL-37-inspired lead peptide SAAP-148 was combined with antibiotics of different classes against Staphylococcus aureus, and showed synergy with teicoplanin. Synergy with teicoplanin was also observed with LL-37, the LL-37-inspired SAAP-276 and the TC-1-inspired TC84. Interestingly, no synergy was observed against Staphylococcus epidermidis. Furthermore, teicoplanin combined with SAAP-148 or SAAP-276 showed strong interaction against S. aureus biofilms. The dltABCD operon and the mprF gene in S. aureus conferred resistance to LL-37, but SAAP-148 proved to be indifferently potent against wild-type, ΔdltA and ΔmprF S. aureus strains. When used alone, relatively high concentrations of both LL-37 and teicoplanin (30-120 µM and 4-32 mg/L, respectively) were required to kill S. aureus. Resistance to LL-37 in S. aureus was overcome by combined use of teicoplanin and LL-37. Thus, teicoplanin potentiates peptide LL-37, enhancing the efficacy of the innate defence, and combining the novel peptides with teicoplanin offers potential for enhanced efficacy of treatment of S. aureus infections, including biofilms.

Evaluation of the bioNexia Legionella Test, Including Impact of Incubation Time Extension, for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine.

In this study, we compared the bioNexia test (bioMérieux, Marcy-l'Étoile, France), a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 in urine, with the BinaxNOW urinary antigen test (Alere, Waltham, Massachusetts, USA). After 15 min of incubation (in accordance with the manufacturers' instructions), the sensitivities and specificities were, respectively, 76.5% and 97.2% for the bioNexia test and 87.1% and 100% for the BinaxNOW test. After a prolonged incubation time of 60 min, the sensitivities and specificities increased to, respectively, 89.4% and 97.2% for the bioNexia test and 91.8% and 100% for the BinaxNOW test. When the tests were read after 15 min, the concentration of discrepant urine samples increased the sensitivities to 94.1% for both tests. In conclusion, we found that although the bioNexia test showed lower sensitivity for the detection of L. pneumophila antigen in nonconcentrated urine compared to the BinaxNOW test, a prolonged incubation time as well as the use of concentrated samples showed comparable sensitivities for both tests.

Combined use of ultrasound-assisted liposuction and limited-incision platysmaplasty for treatment of the aging neck.

BACKGROUND: Many techniques have been introduced for the surgical treatment of the aging neck. In this study the combination therapy of ultrasound-assisted liposuction and limited-incision platysmaplasty for cervicofacial rejuvenation is presented. METHODS: Fifteen female patients (age = 43-75 years) were treated for grade II-III (n = 2), III (n = 6), and grade IV (n = 7) cervicomental angle deformity. The outcome of surgery was retrospectively evaluated by a panel. RESULTS: Ultrasonic energy was applied for an average of 2 min (range = 45 s-6.5 min). The mean aspiration volume was 125 ml. No immediate or delayed adjuvant skin reduction was needed in any of the patients. No complications were encountered in this series. After treatment significant improvement of the cervicomental angle was observed. CONCLUSION: For treatment of all grades of the aging neck we advocate the combination of UAL and limited-incision platysmaplasty. This combination therapy has little morbidity and leads uniformly to significant improvement of the cervicomental angle.

Natural variations in postpartum maternal care in inbred and outbred mice.

The role of maternal care in mediating variation in offspring phenotype has been examined in the rat and demonstrates that mother-infant interactions are critical for inducing long-term changes in behavior. Though phenotypic differences between mice strains are often attributed to genetic factors, the influence of early maternal environment has not been extensively explored. To understand maternal influence on phenotype in mice, we must first explore the nature of differences in behavior. In the present study, we examine aspects of maternal care differentiating mice strains and explore the relationship between postpartum behavior and measures obtained by a standard test of maternal responsivity (Retrieval Test). We compared inbred 129Sv (n=25), C57BL/6J (n=23), and outbred Swiss (n=23) lactating female mice. Swiss females had shorter latencies to retrieve and crouch over pups (P<.01), whereas 129Sv females had shorter latencies to nestbuild (P<.05). Conversely, observations of homecage behavior indicate that 129Sv females nestbuild less frequently. 129Sv females also engaged in very low levels of pup licking/grooming (P<.001) and long periods of nursing/contact (P<.05) with pups compared to C57BL/6J and Swiss females. Temporal analysis suggests that the magnitude of these differences varies both within and between days. No significant correlations were found between any aspect of maternal responsivity and postpartum behavior. These results illustrate that through detailed analysis of maternal behavior in mice, variations between strains can be observed. These variations represent strain specific strategies for promoting growth and survival of offspring during infancy that may also mediate "epigenetic" differences in phenotype in adulthood.

The integrin binding site 2 (IBS2) in the talin rod domain is essential for linking integrin beta subunits to the cytoskeleton.

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in alpha helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this alpha helix, which disrupted the alpha-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to alphaIIbbeta3 integrin in focal adhesions and to inhibit in vitro this association as shown by an alphaIIbbeta3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1(-/-) cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.

Repeated ankle sprains and delayed neuromuscular response: acceleration time parameters.

STUDY DESIGN: A comparative study. OBJECTIVES: To assess whether in subjects with unilateral chronic ankle instability the dynamic reaction time of the affected ankle differs from the healthy ankle and from ankles of a control group. BACKGROUND: Reaction time is an essential element in joint protection against sudden unexpected excessive movement requiring fast and coordinated muscle action. During a sudden ankle inversion movement, a reflex action of the evertor muscles is needed to counteract the movement. Adequate neuromuscular response is crucial and a delayed response could contribute to inversion trauma and subsequently to chronic ankle instability. The isokinetic dynamometer acceleration time (ACC-TIME) provides valuable information on dynamic neuromuscular ability. MATERIAL AND METHODS: Patients with unilateral chronic ankle instability (n = 11) and healthy individuals in a control group (n = 11) were tested on an isokinetic dynamometer during 3 sets of 3 reciprocal inversion/eversion movements of both ankles at 30 degrees/s and 120 degrees/s. Analysis of variance models were used to compare the ACC-TIME of the affected ankle to the unaffected ankle of the same subjects and a control group. RESULTS: For the evertor muscles at 30 degrees/s and 120 degrees/s a significantly prolonged ACC-TIME was found when comparing the affected ankles to the contralateral ankles and both ankles of the control group. For the invertor muscles at 120 degrees/s a significantly prolonged ACC-TIME was found when comparing the affected ankle to the unaffected ankles of patients and those of the control group. CONCLUSIONS: Because the most important evertor muscles are innervated by the fibular nerve, the significantly prolonged ACC-TIME of the affected ankle is consistent with the finding of a lower motor nerve conduction velocity of the fibular nerve after inversion trauma. The results support the concept of a delayed neuromuscular response as an important factor in the etiology of chronic ankle instability.

The significance of angiogenesis in malignant pheochromocytomas.

The purpose of this study was to investigate tumor angiogenesis in a series of benign and malignant pheochromocytomas and to determine whether there is a correlation between angiogenesis and the presence of distant metastases. In this study, the CD31 monoclonal antibody was selected to measure intratumoral microvessel density. Nineteen patients with malignant pheochromocytomas and nineteen patients with benign pheochromocytomas who underwent operation were studied. In order to quantify intratumoral microvessel density, the total number of pixels of CD31-positive reactivity was assessed and expressed as a percentage of the total tissue area in the analyzed field. Analysis of variance revealed a statistically significant correlation between malignancy and intratumoral microvessel density (p = 0.0009). Although there was a considerable variability in the intratumoral microvessel density from tumor to tumor within both the benign and the malignant group, a percentage of more than 28.5% anti-CD31 stained area was found only in malignant tumors. In conclusion, this study shows that the mean intratumoral microvessel density in malignant pheochromocytomas is increased approximately twofold as compared with benign tumors. However, the clinical significance of this prognostic marker is rather weak, because only 4 of the 19 malignant pheochromocytomas had microvessel density higher than this threshold of 28.5%.

Endoscopic nasogastric-jejunal feeding tube placement in critically ill patients.

BACKGROUND: Historically, placement of small bowel nasoenteric feeding tubes in the critically ill patient has been difficult because of lack of bedside fluoroscopy, inadequately designed endoscopic tubes, or failure of the tube to spontaneously pass into the duodenum following placement. METHODS: We followed-up 54 consecutive critically ill patients who had a combined nasogastric-jejunal feeding tube placed at the bedside using a new endoscopic, nonfluoroscopic method of placement. Data were obtained on the placement procedure, outcomes, and complications that followed. RESULTS: Tubes were successfully placed in 94% of the patients in an average time of 12 minutes. Negative outcomes included the following: inadvertent removal by patient or staff (21%), intolerance to tube feeding (14%), clogging (9%), kinking (6%), and cracking at the tube adapter (11%). The duration of the tube following placement ranged from 1 to 42 days, with an average of 9 days. CONCLUSION: The combined tubes were easy to place endoscopically. The endoscopic, nonfluoroscopic method of placing feeding tubes can be performed at the bedside and allows for gastric decompression and enteral feeding to be rapidly and efficiently achieved, which is particularly useful for intubated patients in an intensive care setting. Negative outcomes should decrease by avoidance of inadvertent tube removal and by improved tube maintenance and materials.

Evaluation of the safety of nurse-assisted percutaneous endoscopic gastrostomy.

Percutaneous endoscopic gastrostomy (PEG) procedures have become a common, nonsurgical approach to providing enteral access to patients who are otherwise unable to meet their nutritional needs by mouth. Historically, two physicians have been required to complete this procedure; the first performed the endoscopy while the second helped to position the PEG tube. As a result of constraints on physicians' time and availability, as well as increased medical costs, this process has changed in some settings where the procedure is accomplished by one physician who performs the endoscopy and directly observes a nurse who acts in the role of the second physician. This research study was designed to evaluate the safety of nurse-assisted PEG procedures by comparing the complications of placement (i.e., infection, hemorrhage, perforation, and ileus) with those complications that occur when two physicians perform the procedure. The current standard of care for placing PEG tube was followed. Results in this small sample show that nurse-assisted PEG procedures are as safe as when two physicians perform this procedure.