Intravenous Lipopolysaccharide Research Articles

Leveraging Hypotension Prediction Index to Forecast LPS-Induced Acute Lung Injury and Inflammation in a Porcine Model: Exploring the Role of Hypoxia-Inducible Factor in Circulatory Shock.

Acute respiratory distress syndrome (ARDS) is a critical illness in critically unwell patients, characterized by refractory hypoxemia and shock. This study evaluates an early detection tool and investigates the relationship between hypoxia and circulatory shock in ARDS, to improve diagnostic precision and therapy customization. We used a porcine model, inducing ARDS with mechanical ventilation and intratracheal plus intravenous lipopolysaccharide (LPS) injection. Hemodynamic changes were monitored using an Acumen IQ sensor and a ForeSight Elite sensor connected to the HemoSphere platform. We evaluated tissue damage, inflammatory response, and hypoxia-inducible factor (HIF) alterations using enzyme-linked immunosorbent assay and immunohistochemistry. The results showed severe hypotension and increased heart rates post-LPS exposure, with a notable rise in the hypotension prediction index (HPI) during acute lung injury (p = 0.024). Tissue oxygen saturation dropped considerably in the right brain region. Interestingly, post-injury HIF-2α levels were lower at the end of the experiment. Our findings imply that the HPI can effectively predict ARDS-related hypotension. HIF expression levels may serve as possible markers of rapid ARDS progression. Further research should be conducted on the clinical value of this novel approach in critical care, as well as the relationship between the HIF pathway and ARDS-associated hypotension.

ABCA1 and apoA-I dependent 12-hydroxyeicosatetraenoic acid efflux regulates macrophage inflammatory signaling.

Aberrant high-density lipoprotein (HDL) function is implicated in inflammation-associated pathologies. While HDL ABCA1-mediated reverse cholesterol and phospholipid transport are well described, the movement of pro-/anti-inflammatory lipids has not been explored. HDL phospholipids are the largest reservoir of circulating arachidonic acid-derived oxylipins. Endotoxin-stimulation activates inflammatory cells leading to hydroxyeicosatetraenoic acid (HETE) production, oxylipins which are involved in inflammatory response coordination. Active signaling in the non-esterified (NE) pool is terminated by sequestration of HETEs as esterified (Es) forms and degradation. We speculate that an ABCA1-apoA-I-dependent efflux of HETEs from stimulated cells could regulate intracellular HETE availability. Here we test this hypothesis both in vitro and in vivo. In endotoxin-stimulated RAW-264.7 macrophages preloaded with d8-arachidonic acid we use compartmental tracer modeling to characterize the formation of HETEs, and their efflux into HDL. We found that in response to endotoxin: I) Cellular NE 12-HETE is positively associated with MCP-1 secretion (p<0.001); II) HETE transfer from NE to Es pools is ABCA1-depedent (p<0.001); III) Cellular Es HETEs are transported into media when both apoA-I and ABCA1 are present (p<0.001); IV) The stimulated efflux of HETEs >> arachidonate (p<0.001). Finally, in endotoxin challenged humans (n=17), we demonstrate that intravenous lipopolysaccharide (0.6 ng/kg body weight) resulted in accumulation of 12-HETE in HDL over a 168-hour follow-up. Therefore, HDL can suppress inflammatory responses in macrophages by regulating intracellular HETE content in an apoA-I/ABCA1 dependent manner. The described mechanism may apply to other oxylipins and explain anti-inflammatory properties of HDL. This newly defined HDL property opens new doors for the study of lipoprotein interactions in metabolic diseases.

Exposure to normobaric hypoxia shapes the acute inflammatory response in human whole blood cells in vivo

Cells of the immune defence, especially leukocytes, often have to perform their function in tissue areas that are characterized by oxygen deficiency, so-called hypoxia. Physiological hypoxia significantly affects leukocyte function and controls the innate and adaptive immune response mainly through transcriptional gene regulation via the hypoxia-inducible factors (HIFs). Multiple pathogens including components of bacteria, such as lipopolysaccharides (LPS) trigger the activation of leukocytes. HIF pathway activation enables immune cells to adapt to both hypoxic environments in physiological and inflammatory settings and modulates immune cell responses through metabolism changes and crosstalk with other immune-relevant signalling pathways. To study the mutual influence of both processes in vivo, we used a human endotoxemia model, challenging participants with an intravenous LPS injection post or prior to a 4-h stay in a hypoxic chamber with normobaric hypoxia of 10.5% oxygen. We analysed changes in gene expression in whole blood cells and determined inflammatory markers to unveil the crosstalk between both processes. Our investigations showed differentially altered gene expression patterns of HIF and target genes upon in vivo treatment with LPS and hypoxia. Further, we found evidence for effects of hypoxic priming upon inflammation in combination with immunomodulatory effects in whole blood cells in vivo. Our work elucidates the complex interplay of hypoxic and inflammatory HIF regulation in human immune cells and offers new perspectives for further clinical research.

Effects of positive end-expiratory pressure on pulmonary perfusion distribution and intrapulmonary shunt during one-lung ventilation in pigs A randomized crossover study.

During one-lung ventilation (OLV), positive end-expiratory pressure (PEEP) can improve lung aeration, but might over-distend lung units and increase intrapulmonary shunt. We hypothesized that higher PEEP shifts pulmonary perfusion from the ventilated to the non-ventilated lung, resulting in a U-shaped relationship with intrapulmonary shunt during OLV. In nine anesthetized female pigs, a thoracotomy was performed and intravenous lipopolysaccharide infused to mimic the inflammatory response of thoracic surgery. Animals underwent OLV in supine position with PEEP of 0 cmH2O, 5 cmH2O, titrated to best respiratory system compliance, and 15 cmH2O (PEEP0, PEEP5, PEEPtitr, and PEEP15, respectively, 45 min each, Latin square sequence). Respiratory, hemodynamic, and gas exchange variables were measured. The distributions of perfusion and ventilation were determined by i.v. fluorescent microspheres and computed tomography, respectively. Compared to two lung ventilation, the driving pressure increased with OLV, irrespective of the PEEP level. During OLV, cardiac output was lower at PEEP15 (5.5 ± 1.5 l/min) than PEEP0 (7.6 ± 3 l/min) and PEEP5 (7.4 ± 2.9 l/min; P=0.004), while the intrapulmonary shunt was highest at PEEP0 (PEEP0: 48.1 ± 14.4 %; PEEP5: 42.4 ± 14.8 %; PEEPtitr: 37.8 ± 11.0 %; PEEP15: 39.0 ± 10.7 %; P=0.027). The relative perfusion of the ventilated lung did not differ among PEEP levels (PEEP0: 65.0 ± 10.6 %; PEEP5:68.7 ± 8.7 %; PEEPtitr: 68.2 ± 10.5 %; PEEP15: 58.4 ± 12.8%; P=0.096), but the centers of relative perfusion and ventilation in the ventilated lung shifted from ventral to dorsal, and from cranial to caudal zones with increasing PEEP. In this experimental model of thoracic surgery, higher PEEP during OLV did not shift the perfusion from the ventilated to the non-ventilated lung, thus not increasing intrapulmonary shunt. This study was registered and approved by the Landesdirektion Dresden, Germany (25-5131/496/33).

Intravenous lipopolysaccharide challenge in early- versus mid-lactation dairy cattle. II: The production and metabolic responses

Most immunometabolic research uses mid-lactation (ML) cows. Cows in early lactation (EL) are in a presumed state of immune suppression/dysregulation and less is known about how they respond to a pathogen. Study objectives were to compare the production and metabolic responses to i.v. LPS and to differentiate between the direct effects of immune activation and the indirect effects of illness-induced hypophagia in EL and ML cows. Cows in EL (n = 11; 20 ± 2 DIM) and ML (n = 12; 131 ± 31 DIM) were enrolled in a 2 × 2 factorial design containing 2 experimental periods (P). During P1 (3 d), cows were fed ad libitum and baseline data were collected. At the initiation of P2 (3 d), cows were randomly assigned to 1 of 2 treatments by lactation stage (LS): (1) EL (EL-LPS; n = 6) or ML (ML-LPS; n = 6) cows administered i.v. a single bolus of 0.09 µg LPS/kg of BW; Escherichia coli O55:B5 or (2) pair-fed (PF) EL (EL-PF; n = 5) or ML (ML-PF; n = 6) cows administered i.v. saline. Administering LPS decreased DMI and this was more severe in EL-LPS than ML-LPS cows (34% and 11% relative to baseline, respectively). By design, P2 DMI patterns were similar in the PF groups compared with their LPS counterparts. Milk yield decreased following LPS (42% on d 1 relative to P1) and despite an exacerbated decrease in EL-LPS cows on d 1 (25% relative to ML-LPS), remained similar between LS from d 2 to 3. The EL-LPS cows had increased milk fat content, but no difference in protein and lactose percentages compared with ML-LPS cows. Further, cumulative ECM yield was increased (21%) in EL-LPS compared with ML-LPS cows. During P2, EL-LPS cows had a more intense increase in MUN and BUN than ML-LPS and EL-PF cows. Administering LPS did not cause hypoglycemia in either EL-LPS or ML-LPS cows, but glucose was increased (33%) in EL-LPS compared with EL-PF. Hyperinsulinemia occurred after LPS, and insulin was further increased in ML-LPS than EL-LPS cows (2.2-fold at 12 h peak). During P2, circulating glucagon increased only in EL-LPS cows (64% relative to all other groups). Both EL groups had increased NEFA at 3 and 6 h after LPS from baseline (56%), but NEFA in EL-LPS cows gradually returned to baseline thereafter and were reduced relative to EL-PF until 36 h (50% from 12 to 24 h). Alterations in BHB did not differ between ML groups, but EL-LPS had reduced BHB compared with EL-PF from 24 to 72 h (51%). Results indicate that there are distinct LS differences in the anorexic and metabolic responses to immune activation. Collectively, EL cows are more sensitive to the catabolic effects of LPS than ML cows, but these exacerbated metabolic responses appear coordinated to fuel an augmented immune system while simultaneously supporting milk synthesis.

Venoarterial Extracorporeal Membrane Oxygenation Implementation in Septic Shock Rat Model.

Septic shock, a global health concern, boasts high mortality rates. Research exploring the efficacy of venoarterial extracorporeal membrane oxygenation (VA-ECMO) in septic shock remains limited. Our study aimed to establish a rodent model employing VA-ECMO in septic shock rats, assessing the therapeutic impact of VA-ECMO on septic shock. Nineteen Sprague-Dawley rats were randomly assigned to sham, septic shock, and (septic shock + VA-ECMO; SSE) groups. Septic shock was induced by intravenous lipopolysaccharides, confirmed by a mean arterial pressure drop to 25-30% of baseline. Rats in the SSE group received 2 hours of VA-ECMO support and 60 minutes of post-weaning ventilation. Sham and septic shock groups underwent mechanical ventilation for equivalent durations. Invasive mean arterial pressure monitoring, echocardiographic examinations, and blood gas analysis revealed the efficacy of VA-ECMO in restoring circulation and ensuring adequate tissue oxygenation in septic shock rats. Post-experiment pathology exhibited the potential of VA-ECMO in mitigating major organ injury. In summary, our study successfully established a stable septic shock rat model with the implementation of VA-ECMO, offering a valuable platform to explore molecular mechanisms underlying VA-ECMO's impact on septic shock.

Intravenous lipopolysaccharide challenge in early- versus mid-lactation dairy cattle. I: The immune and inflammatory responses

Cows in early lactation (EL) are purportedly immune suppressed, which renders them more susceptible to disease. Thus, the study objective was to compare key biomarkers of immune activation from i.v. LPS between EL and mid-lactation (ML) cows. Multiparous EL (20 ± 2 DIM; n = 11) and ML (131 ± 31 DIM; n = 12) cows were enrolled in a 2 × 2 factorial design and assigned to 1 of 2 treatments by lactation stage (LS): (1) EL (EL-LPS; n = 6) or ML (ML-LPS; n = 6) cows administered a single LPS bolus from Escherichia coli O55:B5 (0.09 µg/kg of BW), or (2) pair-fed (PF) EL (EL-PF; n = 5) or ML (ML-PF; n = 6) cows administered i.v. saline. After LPS administration, cows were intensely evaluated for 3 d to analyze their response and recovery to LPS. Rectal temperature increased in LPS relative to PF cows (1.1°C in the first 9 h), and the response was more severe in EL-LPS relative to ML-LPS cows (2.3 vs. 1.3°C increase at 4 h post-LPS; respectively). Respiration rate increased only in EL-LPS cows (47% relative to ML-LPS in the first hour post-LPS). Circulating tumor necrosis factor-α, IL-6, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and IFN-γ-inducible protein-10 increased within the first 6 h after LPS and these changes were exacerbated in EL-LPS relative to ML-LPS cows (6.3-fold, 4.8-fold, 57%, 93%, 10%, and 61%, respectively). All cows administered LPS had decreased circulating iCa relative to PF cows (34% at the 6 h nadir), but the hypocalcemia was more severe in EL-LPS than ML-LPS cows (14% at 6 h nadir). In response to LPS, neutrophils decreased regardless of LS, then increased into neutrophilia by 24 h in all LPS relative to PF cows (2-fold); however, the neutrophilic phase was augmented in EL- compared with ML-LPS cows (63% from 24 to 72 h). Lymphocytes and monocytes rapidly decreased then gradually returned to baseline in LPS cows regardless of LS; however, monocytes were increased (57%) at 72 h in EL-LPS relative to ML-LPS cows. Platelets were reduced (46%) in LPS relative to PF cows throughout the 3-d following LPS, and from 24 to 48 h, platelets were further decreased (41%) in EL-LPS compared with ML-LPS. During the 3-d following LPS, serum amyloid A (SAA), LPS-binding protein (LBP), and haptoglobin (Hp) increased in LPS compared with PF groups (9-fold, 72%, and 153-fold, respectively), and the LBP and Hp responses were more exaggerated in EL-LPS than ML-LPS cows (85 and 79%, respectively) whereas the SAA response did not differ by LS. Thus, our data indicates that EL immune function does not appear "suppressed," and in fact many aspects of the immune response are seemingly functionally robust.

Using a wearable patch to develop a digital monitoring biomarker of inflammation in response to LPS challenge.

Remote inflammation monitoring with digital health technologies (DHTs) would provide valuable information for both clinical research and care. Controlled perturbations of the immune system may reveal physiological signatures which could be used to develop a digital biomarker of inflammatory state. In this study, molecular and physiological profiling was performed following an invivo lipopolysaccharide (LPS) challenge to develop a digital biomarker of inflammation. Ten healthy volunteers received an intravenous LPS challenge and were monitored for 24 h using the VitalConnect VitalPatch (VitalPatch). VitalPatch measurements included heart rate (HR), heart rate variability (HRV), respiratory rate (RR), and skin temperature (TEMP). Conventional episodic inpatient vital signs and serum proteins were measured pre- and post-LPS challenge. The VitalPatch provided vital signs that were comparable to conventional methods for assessing HR, RR, and TEMP. A pronounced increase was observed in HR, RR, and TEMP as well as a decrease in HRV 1-4 h post-LPS challenge. The ordering of participants by magnitude of inflammatory cytokine response 2 h post-LPS challenge was consistent with ordering of participants by change from baseline in vital signs when measured by VitalPatch (r = 0.73) but not when measured by conventional methods (r = -0.04). A machine learning model trained on VitalPatch data predicted change from baseline in inflammatory protein response (R2 = 0.67). DHTs, such as VitalPatch, can improve upon existing episodic measurements of vital signs by enabling continuous sensing and have the potential for future use as tools to remotely monitor inflammation.

Commonly disrupted pathways in brain and kidney in a pig model of systemic endotoxemia

Sepsis is a life-threatening state that arises due to a hyperactive inflammatory response stimulated by infection and rarely other insults (e.g., non-infections tissue injury). Although changes in several proinflammatory cytokines and signals are documented in humans and small animal models, far less is known about responses within affected tissues of large animal models. We sought to understand the changes that occur during the initial stages of inflammation by administering intravenous lipopolysaccharide (LPS) to Yorkshire pigs and assessing transcriptomic alterations in the brain, kidney, and whole blood. Robust transcriptional alterations were found in the brain, with upregulated responses enriched in inflammatory pathways and downregulated responses enriched in tight junction and blood vessel functions. Comparison of the inflammatory response in the pig brain to a similar mouse model demonstrated some overlapping changes but also numerous differences, including oppositely dysregulated genes between species. Substantial changes also occurred in the kidneys following LPS with several enriched upregulated pathways (cytokines, lipids, unfolded protein response, etc.) and downregulated gene sets (tube morphogenesis, glomerulus development, GTPase signal transduction, etc.). We also found significant dysregulation of genes in whole blood that fell into several gene ontology categories (cytokines, cell cycle, neutrophil degranulation, etc.). We observed a strong correlation between the brain and kidney responses, with significantly shared upregulated pathways (cytokine signaling, cell death, VEGFA pathways) and downregulated pathways (vasculature and RAC1 GTPases). In summary, we have identified a core set of shared genes and pathways in a pig model of systemic inflammation.

Inflammatory Response to Intravenously Administered Lipopolysaccharide (LPS) in the Bone Marrow of Healthy Volunteers As a Model for Early Phase Drug Development

Introduction Inflammatory responses in the peripheral and central hematological compartments are important for identification of new biomarkers and drug targets in early phase drug development. There is an increasing interest to investigate the modulation of the inflammatory response upon experimental drug exposure using cellular and molecular analyses, specifically in the central compartment. The lipopolysaccharide (LPS) challenge model allows to study TLR4-mediated local and systemic inflammation and therefore the effects of anti-inflammatory compounds. In addition, there is an interest in the effects of TLR4 agonists on early immune cell populations. The aim of the current study is to investigate the effects of intravenous (i.v.) LPS on the peripheral blood and central bone marrow compartment and to perform a proof-of-methodology study for bone marrow analysis in early phase drug research in healthy volunteers. Methods This was an open-label study in 13 healthy young male subjects, aged 18 - 35 years (inclusive). The study was conducted at the Centre for Human Drug Research, Leiden, The Netherlands. The study protocol was approved by the Medical Ethics Committee. All subjects provided written informed consent prior to any study activity. Subjects were divided into control group (n=3), 1 ng/kg i.v. LPS group (n=5) and 2 ng/kg i.v. LPS group (n=5). Blood and bone marrow samples were collected at baseline and at 4h post-dose timepoints. Primary objective was to evaluate the safety and tolerability of bone marrow sampling in healthy volunteers, by continuous safety monitoring, visual analogue scale (VAS) for pain and subject experience questionnaires. To investigate the pharmacodynamic (PD) effects of i.v. LPS challenge on the bone marrow and whole-blood immune compartments, flow cytometry, cytokine analysis and RNA sequencing was performed. Results All 13 subjects completed the study. Adverse events were mild and reversible, and included puncture site pain related to bone marrow puncture, and flu-like symptoms related to LPS administration. Twelve subjects (92%) indicated they would undergo the procedure again in the future. There was an observable difference in VAS pain score between the baseline bone marrow puncture and LPS treatment groups, but no difference between 1 ng/kg and 2 ng/kg groups. Phenotypic changes of inflammatory signatures in peripheral blood and bone marrow cell populations upon LPS administration were detected by multi-parameter flow cytometry. An LPS-induced inflammatory cytokine profile was present. RNA sequencing revealed differential gene expression in inflammatory gene signatures between peripheral blood mononuclear cells and bone marrow cells. Conclusion We demonstrated that bone marrow analysis is feasible and well-tolerable in early phase studies with healthy volunteers and can be used to assess inflammatory responses in the central hematological compartment upon systemic LPS challenge. Access to bone marrow can provide important novel insights into drug effects on the hematopoiesis and differentiation of immune cells and provides added value to early drug development.

103 Development of a Novel, Experimental, Minimally Invasive Model to Investigate the Genesis and Etiology of Liver Abscesses in Cattle

Abstract Most research surrounding liver abscesses involves post-hoc evaluation of naturally occurring abscesses in production settings. Few studies have been designed to experimentally induce abscesses with the purpose of identifying a time course, causality, and associated physiological implications. Therefore, we set out to develop a minimally invasive, reliable, and repeatable model to induce liver abscesses in calves in a controlled research environment to elucidate the genesis and etiology of the disease, with the ultimate goal of developing abscess reduction strategies. Experiment 1 was an attempt to induce abscesses by intraruminal inoculation with Fusobacterium necrophorum, Salmonella lubbock, and Truperella pyogenes (FST) following an intravenous lipopolysaccharide challenge. Holstein steers were harvested 3- and 10-d after inoculation, but no abscesses were detected. Experiments 2 and 3 involved diet manipulation by cycling calves on and off a high-starch, acidotic diet followed by intraruminal inoculation of microbes. In Experiment 2, Holstein calves were fed a negative control diet, an acidotic diet, and an acidotic diet plus bacterial inoculation with FST. No abscesses formed in the control or acidotic diet calves, but 43% of calves fed the acidotic diet with FST inoculation formed liver abscesses, suggesting that acidosis alone is insufficient to produce abscesses. In Experiment 3, treatments consisted of Holstein steers fed a negative control, an acidotic diet, an acidotic diet plus ruminal Fusobacterium necrophorum (F), and an acidotic diet plus Fusobacterium and Salmonella (FS). Again, there were no abscesses in the control or calves fed an acidotic diet alone; however, 40% of calves receiving F presented with an abscess, and 50% of the calves that received FS presented with abscesses. These data suggest that ruminal acidosis in conjunction with a ruminal load of pathogens commonly associated with liver abscesses provides a model that will induce liver abscesses. While the role of barrier dysfunction in the rumen and the lower gastrointestinal tract in formation of abscesses is not fully understood, this work provides a foundation to further explore the etiology of this disease and potential mitigation strategies.

Polb 001, an Oral Broad-Spectrum Anti-Inflammatory with the Potential to Prevent Cytokine Release Syndrome (CRS)

Background Cytokine Release Syndrome (CRS) is a well-recognized toxicity that occurs with high frequency following treatments such as T-cell engaging antibodies, Chimeric Antigen Receptor (CAR) T cells and other T-cell boosting immunotherapies. In addition to patient mortality and morbidity, the high frequency of CRS associated with these treatments represents a barrier to delivery in the outpatient setting. The need for inpatient management of CRS adds to the costs of these treatments and contributes to restricting their availability. Current treatments are dominated by reactive administration of tocilizumab, steroids or anakinra. POLB 001 is a p38 MAPK inhibitor in development for severe influenza infections and CRS. In vivo, POLB 001 acts as a broad-spectrum anti-inflammatory by potently inhibiting the expression of proinflammatory cytokines such as IL-1β, IL-6, IL-8, IL-10, TNFα, COX-2. Methods In this single-center, randomized, double blinded, placebo-controlled, multiple dose study (NCT05765955), 36 healthy volunteers were orally administered 30, 70 or 150 mg POLB 001 or placebo BD (3:1 active to placebo) for 7 days. Each subject was then challenged with intradermal lipopolysaccharide (LPS) and intravenous LPS which drive local and systemic inflammation, respectively. The study objectives were to study the effect of POLB 001 on inflammatory responses following LPS challenge, including cytokine analysis, exploratory analysis including PK and metabolite profile and the safety and tolerability of POLB 001. Results No deaths or serious adverse events occurred during the study. The most commonly reported AE was headache, with the highest number of participants and events in the placebo group (8 (88.9%) subjects and 14 events) followed by the POLB 001 30 mg group (6 (66.7%) subjects and 11 events). Other reported AEs included chills (also more frequent in placebo group) and dizziness (placebo, POLB001 30, 70, 150 mg groups, frequency 22.2%, 11.1%, 33.3%, 22.2%, respectively). POLB 001 reduced levels of activated p38 (phospho-p38) in circulating monocytes by 32.0-60.9%. Reductions were seen in all cytokines measured. Significant reductions were seen in plasma IL-6, IL-8, IL-10 and TNFα. IL-1β levels were below the LLOQ in active and placebo groups. LPS-driven C-Reactive Protein (CRP) was reduced by 70 and 150 mg doses of POLB 001 (33.1 and 33.3%, respectively). Intravenous LPS challenge elevated body temperature by 1°C and HR by &amp;gt;25 BPM with a peak 3 hours following infusion. POLB 001 significantly reduced HR elevations with an estimated difference in peak HR of -8.8 and -7.8 in the 70 and 150 mg groups respectively. Conclusion POLB 001 is safe and tolerable in healthy volunteers. In a human LPS challenge model driving an acute systemic inflammatory response, POLB 001 showed anti-inflammatory effects and clinically meaningful parameters of inflammation were also reduced. The results support further clinical development of POLB 001 for inflammatory diseases and CRS. POLB 001 is undergoing preclinical evaluation in humanized mouse models (NSG-(KbDb)null (IA)null) of T-cell engaging bispecific antibodies, CD19 CAR-T cell therapy for hematological malignancies and for CD28 induced cytokine storm. A Phase 2 trial of POLB 001 in Multiple Myeloma patients receiving T-cell engaging antibodies is planned.

Study on the antipyretic mechanism of large pushing Tianheshui for young rabbits with endotoxin-induced fever

ObjectiveTo observe the effect of large pushing Tianheshui manipulation intervention on the body temperature of young rabbits with endotoxin-induced fever and discuss its antipyretic mechanism.MethodsThirty-two young rabbits meeting the standards were selected from 40 ordinary young male New Zealand rabbits after being adapted for 7 d, and randomly divided into a normal group, a model group, a large pushing Tianheshui group, and an ibuprofen group according to the random number table method, with 8 rabbits in each group. Rabbits in the model group, the large pushing Tianheshui group, and the ibuprofen group were subjected to preparing the endotoxin-induced fever model by intravenous lipopolysaccharide from the marginal ear vein. Rabbits in the large pushing Tianheshui group received Tuina (Chinese therapeutic massage) manipulation intervention 1.5 h and 2.5 h after modeling, respectively. Rabbits in the ibuprofen group were intragastrically given ibuprofen suspension 1.5 h after modeling. The dynamic changes in body temperature were observed for the young rabbits after fever modeling. Enzyme-linked immunosorbent assay was used to determine the content changes in positive mediators of hypothalamic body temperature, including prostaglandin (PG) E2 and cyclic adenosine monophosphate (cAMP), as well as negative mediators of hypothalamic body temperature, including arginine vasopressin (AVP) and α-melanocyte stimulating hormone (α-MSH).ResultsThe body temperature of the young rabbits in the model group was significantly higher than that in the normal group at 0.5–4.0 h, 5.0 h, and 5.5 h after modeling (P<0.01), showing two obvious fever peaks in the model group at 1.5 h and 3.0 h after modeling, respectively, with the highest peak at 1.5 h. Compared with the model group, body temperatures of the large pushing Tianheshui group and the ibuprofen group decreased significantly after 0.5 h of intervention (P<0.05). Compared with the normal group, the contents of PGE2 and cAMP were significantly increased (P<0.05), and the contents of AVP and α-MSH were significantly decreased (P<0.01) in the hypothalamus of the model group. Compared with the model group, the contents of PGE2 and cAMP were significantly decreased (P<0.01), and the levels of AVP and a-MSH were significantly increased (P<0.01) in the hypothalamus in the large pushing Tianheshui group and the ibuprofen group. There were no significant differences in the PGE2, cAMP, AVP, and α-MSH contents in the hypothalamus between the ibuprofen group and the large pushing Tianheshui group (P>0.05).ConclusionLarge pushing Tianheshui manipulation has a significant antipyretic effect on endotoxin-induced fever in young rabbits. The mechanism may be related to inhibiting the positive regulators (PGE2 and cAMP) and promoting the negative regulators (AVP and α-MSH) of hypothalamic control of body temperature.

O39 | Effects of flunixin meglumine on the systemic inflammatory response to intravenous lipopolysaccharide in horses

Journal of Veterinary Pharmacology and TherapeuticsVolume 46, Issue S1 p. 46-47 ORAL PRESENTATIONS O39 | Effects of flunixin meglumine on the systemic inflammatory response to intravenous lipopolysaccharide in horses First published: 11 June 2023 https://doi.org/10.1111/jvp.13205Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, IssueS1Special Issue: 15th International Congress of the European Association for Veterinary Pharmacology and Toxicology held Bruges, Belgium, July 2–5, 2023June 2023Pages 46-47 RelatedInformation

Eucalcemia during lipopolysaccharide challenge in postpartum dairy cows: I. Clinical, inflammatory, and metabolic response

Hypocalcemia induced by immune activation is a conserved response across mammalian species; however, administration of Ca is discouraged in other species as it is associated with increased morbidity and mortality. Early postpartum cows experience a decrease in circulating Ca concentration following acute inflammation. Corrective Ca therapy during the transition period, particularly in dairy cows experiencing acute disease, is common practice. However, the effect of Ca administration on the inflammatory response during acute immune activation is unknown. Our objective was to compare the clinical, inflammatory, and metabolic response to an intravenous (IV) lipopolysaccharide (LPS) challenge between postpartum cows infused, or not, with IV Ca to maintain eucalcemia. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) or sterile 0.9% NaCl (CTRL) during an IV LPS challenge (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). Ionized Ca (iCa) was monitored cow-side, and IV Ca infusion was adjusted in a eucalcemic clamp for 12 h following the start of LPS infusion. Cows were monitored during the 24 h following challenge and serial blood samples were collected to quantify concentrations of glucose, β-hydroxybutyrate, nonesterified fatty acids, urea nitrogen, cytokines, acute-phase proteins, and cortisol. Blood iCa concentration decreased to 0.87 ± 0.03 mM in CTRL during challenge, and by design, iCa concentration was maintained within 3% of baseline in IVCa. Body temperature, heart rate, and respiratory rate were monitored for 24 h following the start of challenge and did not differ between groups. A treatment × time interaction was identified such that serum cortisol concentrations increased in both groups at 2 h but decreased to a greater extent at 6 h in IVCa compared with CTRL. Rumination time (min/h) over the first 12 h following challenge was greater in IVCa, but total rumination time in the 24 h following challenge did not differ from CTRL. Serum glucose and nonesterified fatty acid concentrations decreased, and β-hydroxybutyrate and urea nitrogen concentrations increased over time, but did not differ between groups. Acute leukopenia occurred in both groups at 4 h before leukocytosis was observed at 24 h with total white blood cell counts returning to baseline within 72 h. Plasma concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) increased within 1 h following the start of challenge and did not differ between groups. Serum haptoglobin and serum amyloid A concentrations increased within the 24 h following challenge and were elevated through 72 h but did not differ between groups. Eucalcemia during the acute systemic inflammatory response did not alter the TNF or IL-10 cytokine response, or the acute-phase protein SAA and haptoglobin response in this LPS challenge model; however, eucalcemia was associated with a more rapid decline in cortisol response and greater rumination time in the first 12 h following challenge. We did not find evidence that eucalcemia exacerbated the inflammatory response in early postpartum cows, but Ca administration may alter the clinical response to acute systemic inflammation.

Combining accelerometers and direct visual observations to detect sickness and pain in cows of different ages submitted to systemic inflammation

Cattle suffering from inflammatory infection display sickness and pain-related behaviours. As these behaviours may be transient and last only a few hours, one may miss them. The aim of this study was to assess the benefit of combining continuous monitoring of cow behaviour via collar-attached accelerometers with direct visual observations to detect sickness and pain-related behavioural responses after a systemic inflammatory challenge (intravenous lipopolysaccharide injection) in cows of two different ages, proven by clinical, physiological and blood parameters. Twelve cloned Holstein cows (six ‘old’ cows aged 10–15 years old and six ‘young’ cows aged 6 years old) were challenged and either directly observed at five time-points from just before the lipopolysaccharide injection up to 24 h post-injection (hpi) or continuously monitored using collar-attached accelerometers in either control or challenge situations. Direct observations identified specific sickness and pain behaviours (apathy, changes in facial expression and body posture, reduced motivation to feed) expressed partially at 3 hpi and fully at 6 hpi. These signs of sickness and pain behaviours then faded, and quicker for the young cows. Accelerometers detected changes in basic activities (low ingesting, low ruminating, high inactivity) and position (high time standing up) earlier and over a longer period of time than direct observations. The combination of sensors and direct observations improved the detection of behavioural signs of sickness and pain earlier on and over the whole study period, even when direct signs were weak especially in young cows. This system could provide great benefit for better earlier animal care.

TLR4 and MD2 variation among horses with differential TNFα baseline concentrations and response to intravenous lipopolysaccharide infusion

Gram-negative bacterial septicemia is mediated through binding of lipopolysaccharide (LPS) to mammalian toll-like receptor protein 4 (TLR4). TLR4 and its cognate protein, myeloid differentiation factor 2 (MD2) form a heterodimeric complex after binding LPS. This complex induces a cascade of reactions that results in increased proinflammatory cytokine gene expression, including TNFα, which leads to activation of innate immunity. In horses, the immune response to LPS varies widely. To determine if this variation is due to differences in TLR4 or MD2, DNA from 15 healthy adult horses with different TNFα dynamics after experimental intravenous LPS infusion was sequenced across exons of TLR4 and MD2. Haplotypes were constructed for both genes using all identified variants. Four haplotypes were observed for each gene. No significant associations were found between either TNFα baseline concentrations or response to LPS and haplotype; however, there was a significant association (P value = 0.0460) between the baseline TNFα concentration and one MD2 missense variant. Three-dimensional structures of the equine TLR4-MD2-LPS complex were built according to haplotype combinations observed in the study horses, and the implications of missense variants on LPS binding were modeled. Although the sample size was small, there was no evidence that variation in TLR4 or MD2 explains the variability in TNFα response observed after LPS exposure in horses.

An updated profile of the bovine acute phase response following an intravenous lipopolysaccharide challenge.

The objective was to provide an updated profile of the bovine acute-phase response to include recent advancements in technologies and expanded hematological, cytokine, and serum chemistry variables. Beef steers (n = 32; body weight [BW] = 251 ± 19.5 kg) were fitted with indwelling jugular catheters 1 d before lipopolysaccharide (LPS; 0.25 µg LPS/kg BW from Escherichia coli O111:B4) administration to facilitate serial blood collection. Rectal temperature was measured using indwelling probes, and ocular temperature was measured using infrared thermal imaging. Blood samples were collected for subsequent analysis of serum chemistry, hematology, and cytokine concentrations. Pearson correlation of rectal temperature and ocular infrared temperature was 0.61 (P < 0.01) and the Spearman correlation coefficient was 0.56 (P < 0.01). Interactions of hour × method were observed for ocular and rectal measurements of body temperature in response to endotoxin exposure. Maximum observed temperature was 39.6 °C at 2.5 h for both rectal and ocular measurements. Body temperature differed by method at hours 0.5, 2.5, 4.5, 7.5, 12.5, 36.5, and 47.5 (P < 0.01), but were not different otherwise. All variables of serum chemistry and complete blood count were influenced by LPS administration, except creatinine, serum glucose, and percent basophils (P ≤ 0.02). Alanine aminotransferase and alkaline phosphatase peaked at hour 2 relative to LPS administration, returned to baseline at hour 12 and continued to decrease below the baseline value at hour 48 (P < 0.01). Total protein concentration decreased 3% in response to LPS (P = 0.01). Total white blood cell count decreased 75% after LPS administration at hour 1 (P < 0.01). Lymphocyte count recovered to baseline at hour 6; sooner than neutrophil count at hour 36. Serum cortisol concentration increased 294% relative to baseline at hour 1 followed by a sustained decrease and return to normal concentration at hour 4 (P < 0.01). Additionally, circulating cytokine concentrations changed with time in response to the LPS challenge, excluding aFGF, bFGF, IGF-1, IL-2, IL-4, MCP-1, and ANG-1 (P ≤ 0.08). Maximum observed concentration of TNF-α at hour 1 was 117% greater than the pre-challenge value (P < 0.01). Data presented herein add to existing works to understand the endocrine and immune responses of beef steers administered exogenous LPS, and incorporate recent technologies, additional biomarkers, and an expanded cytokine profile that can be used as referential data in future research.

Platelet TLR9 Promotes Pulmonary Thrombo-Inflammation in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder, which affects millions of people worldwide. Erythrocyte sickling, chronic hemolysis and ischemia-reperfusion injury promote sterile inflammation in SCD.Recent evidence suggests that activation of innate immune pathways in platelets contribute to development of acute chest syndrome (ACS), a type of acute lung injury in SCD by promoting occlusion of lung arterioles by neutrophil-platelet-erythrocyte aggregates (pulmonary thrombo-inflammation). Although, TLR9 is functionally expressed on the surface of activated platelets and may bind to circulating double stranded DNA, the role of platelet TLR-9 in promoting pulmonary thrombo-inflammation in SCD remains elusive. Our initial findings using intravenous lipopolysaccharide (0.1 μg/kg LPS) or oxy-hemoglobin (10 μmole/kg oxy-Hb) triggered model of vaso-occlusive crisis in transgenic-humanized SCD mice reveal that pulmonary vaso-occlusion is associated with elevated surface-expression of platelet TLR-9 in SCD mice. Unlike SCD mice, surface-expression of TLR9 was undetectable in the platelets of control mice challenged with either IV LPS or oxy-Hb. These findings suggest a potential role for platelet TLR9 in promoting lung vaso-occlusion and injury in SCD. Currently, studies are in progress to identify the innate immune signaling downstream to platelet TLR9, contributing to pulmonary thrombo-inflammation in SCD.

Quercetin Supplement to Aspirin Attenuates Lipopolysaccharide-Induced Pre-eclampsia-Like Impairments in Rats Through the NLRP3 Inflammasome.

Aspirin is a common drug for the treatment of pre-eclampsia. We aimed to explore whether quercetin as a supplement to aspirin could enhance the therapeutic outcome in pre-eclampsia rat models. We further aimed to evaluate the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome as a potential pre-eclampsia-related molecular mechanism, which can be affected by quercetin treatment. Rat pre-eclampsia models were established using an intravenous lipopolysaccharide injection after gestation. Rats were treated with aspirin and quercetin at 6-18days after pregnancy. On day 20, blood, fetus, and placenta were harvested. Blood pressure and the level of proteinuria were measured every 4days. Fetal outcomes were analyzed by pup body weight. Serum soluble Fms-like tyrosine kinase-1, PIGF, interleukin-6, and interleukin-10 levels were measured using the enzyme-linked immunosorbent assay. Caspase-1, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and p-caspase-1 levels in the placenta were assessed using western blot or quantitative real-time polymerase chain reaction analyses. Pre-eclampsia rat models showed a pronounced increase in systolic blood pressure and proteinuria after 4days of pregnancy, while aspirin, quercetin, and aspirin/quercetin combinatory treatment significantly attenuated the blood pressure and proteinuria abnormalities. Notably, the aspirin/quercetin combinatory treatment showed the highest efficacy in attenuating pre-eclampsia-like symptoms. Placental caspase-1 and NLRP3 levels also showed the greatest attenuation in pre-eclampsia rats after aspirin/quercetin treatment. Our data suggested that quercetin supplementation to aspirin is more effective in attenuating symptoms of pre-eclampsia and improving pregnancy outcomes compared with quercetin or aspirin alone. Quercetin can ameliorate placental NLRP3 inflammasome activation, which might serve as an underlying mechanism for its therapeutic efficacies in pre-eclampsia.