Colony-stimulating Factor Concentrations Research Articles

73 Effects of blend of organic acids and monoglycerides on immune responses of weaned pigs experimentally infected with enterotoxigenic Escherichia coli F18

Abstract Our previous research implies that dietary supplementation of organic acids blend, monoglycerides blend, and the combination of both can mitigate diarrhea of weaned pigs infected with Escherichia coli (E. coli) F18. The objective of this study was to investigate the effects of dietary supplementation with a blend of organic acids, a blend of monoglycerides, or the combination of both on immune responses of weaned pigs experimentally infected with E. coli F18. Forty pigs [average body weight (BW) = 7.81 ± 0.84 kg] were individually housed and randomly assigned to one of four treatment groups with 10 replicate pigs per treatment. The four dietary treatments were: () Control: nursery basal diet, 2) Acids: basal diet supplemented with a blend of organic acids (formic acid, lactic acid, and sodium formate) at 0.3%, 3) Monoglycerides: basal diet supplemented with a blend of monoglycerides of fatty acids at 0.3%, or 4) Combination of acids and monoglycerides: basal diet added with the acids blend at 0.2% and the monoglycerides blend at 0.2%. The experiment lasted 28 d, with 7 d before and 21 d after the first inoculation (d 0). All pigs were orally inoculated with E. coli F18 (1010 CFU/3 mL) for 3 consecutive days from d 0. Serum samples were collected on d 0, 2, 5, and 14 post-inoculation (PI) to measure the concentration of C-reactive protein (CRP) and haptoglobin using ELISA. Porcine Cytokine/Chemokine13-Plex Discovery Assay (Eve Technology, Canada) was also conducted with serum samples from d 0, 5, and 14 PI. Data were analyzed by ANOVA using PROC MIXED of SAS with a randomized complete block design. The concentrations of serum CRP and haptoglobin were increased (P < 0.0001) on d 2 PI and then gradually decreased on d 5 and 14 PI. However, no differences were observed in serum CRP and haptoglobin concentrations among all treatment groups throughout the experiment. Pigs fed with monoglycerides tended (P < 0.10) to have decreased serum granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1-alpha (IL-1α), and tumor necrosis factor-alpha (TNF-α) concentrations on d 0 PI compared with other treatments. Pigs supplemented with monoglycerides also tended (P < 0.10) to have decreased GM-CSF on d 5 PI and less IL-1α on d 14 PI among all treatments. Supplementation of the combination tended (P < 0.10) to have greater GM-CSF concentration on d 5 PI compared with other treatments. In conclusion, dietary supplementation of monoglycerides blend or the combination of organic acids and monoglycerides alleviates systemic inflammation of weaned pigs during E. coli F18 infection by modulating related cytokines. However, additional research is needed to understand the modulation mechanisms.

Abstract 6642: Exploring the x-ray psoralen activated cancer therapy mechanism of action in a genetically engineered mouse model of soft tissue sarcoma

Abstract BACKGROUND: Psoralen is a natural ultraviolet activated compound that has been used for over 70 years to treat skin cancer, psoriasis, and vitiligo. Recently, to expand its use beyond external treatment, a novel therapeutic called X-PACT (X-ray psoralen activated cancer therapy) was created to treat internal tumors. A combination of 8-methoxypsoralen and phosphors that convert low doses of x-ray radiation to ultraviolet (UV) light are injected into solid tumors and subsequently activated by x-ray photons with considerable antineoplastic effect. Notably, canine clinical trials have observed significant abscopal effects of X-PACT therapy on metastatic lesions throughout patients, but the immunomodulatory mechanisms of action are unclear (Nolan et al, in submission). Here, we present preliminary data investigating the immune response to X-PACT therapy in a murine soft tissue sarcoma (STS) model. METHODS: Six mice of the inducible KrasG12D/+; p53flox/flox (KP) murine STS model with palpable, gastrocnemius tumors were used in this initial study. Four mice underwent treatment with X-PACT. In this group, psoralen was mixed with energy converters and injected into the tumor. The mice then received low-level radiation to activate the drug. Treatment was performed five times over a 15-day period and the animals were sacrificed within 48 hours of final treatment. The other two mice with induced tumors received no treatment prior to sacrifice. For both groups, a cardiac blood draw was performed at the time of sacrifice to obtain plasma serum for analysis. Luminex xMAP technology was used for multiplexed quantification of 45 mouse cytokines, chemokines and growth factors. RESULTS: There were five markers that showed significantly different levels between the treated and control groups. Results indicated significantly higher (p = 0.006) plasma concentrations of macrophage derived chemokine (MDC) following five rounds of treatment with X-PACT therapy. Additionally, the treated mice displayed significantly lower concentrations of granulocyte colony-stimulating factor (G-CSF, p = 0.003), eotaxin (p = 0.016), tissue inhibitor of matrix metalloproteinase 1 (TIMP1, p = 0.024), and interleukin-1β (IL-1β, p = 0.033) compared to untreated mice with STS. CONCLUSION: In humans, MDC levels are associated with longer survival and lower cancer severity. G-CSF, eotaxin, TIMP1, and IL-1β are upregulated in cancer patients and correlate with disease severity. Together, the lower concentrations of G-CSF, Eotaxin, TIMP1, and IL-1β suggest a softening of cancer severity following X-PACT treatment. Further studies and larger sample sizes are necessary to confirm and expand upon these preliminary findings. Citation Format: Beatrice Schleupner, Alexandra Hunter Aitchison, Elle MacAlpine, Nicole Cantor, Jason Somarelli, Brian Brigman, Julia Visgauss, Michael Monument, William Eward. Exploring the x-ray psoralen activated cancer therapy mechanism of action in a genetically engineered mouse model of soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6642.

Effect of bromelain on periodontal destruction and alveolar bone in rats with experimental periodontitis.

Several substances that have anti-inflammatory, antiproteinase, and anti-infective properties have been evaluated as modulators of the inflammatory response in periodontal disease. However, evidence for the anti-inflammatory and antioxidative activities of bromelain is limited. This study evaluated the impact of systemically administered bromelain on the progression of experimental periodontitis. Four equal groups of 32 Wistar albino rats were created as follows (n=8): control, periodontitis+saline, periodontitis+5mg/kg/day bromelain, and periodontitis+10mg/kg/day bromelain. To quantify the resorption of bone and bone volume/tissue volume, bone surface / bone volume, and connectivity, lower jawbones were fixed and then scanned using microcomputed tomography (micro CT). Blood samples were taken to measure the macrophage colony-stimulating factor(M-CSF) concentrations, receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase-8 (MMP-8), interleukin-6(IL-6), glutathione peroxidase (GPx), superoxide dismutase (SOD), and malondialdehyde (MDA). Histopathological assessments were made to examine the tissue. Treatment with bromelain improved the healing of the periodontium by decreasing the number of leukocytes and ligament deterioration in the gingival connective tissue and by supporting reintegration with alveolar bone. Bromelain used in ligature-induced periodontitis reduced alveolar bone (AB) resorption as measured by microCT; reduced inflammatory parameters such as IL-6 and TNF-α; regulated oxidative-antioxidative processes by increasing GPx and SOD and reducing MDA levels; and regulated AB modeling by decreasing M-CSF, RANKL, and MMP-8 and increasing OPG levels. Bromelain may be an option in periodontal therapy by regulating cytokine levels, improving the healing process, and reducing bone resorption and oxidative stress.

Anxiety-like Behavior in Female Sprague Dawley Rats Associated with Cecal Clostridiales.

The relationship between the microbiota profile and exposure to stress is not well understood. Therefore, we used a rat model of unpredictable chronic mild stress (UCMS) to investigate this relationship. Depressive-like behaviors were measured in Female Sprague Dawley rats using the sucrose preference test and the Porsolt swim test. Anxiety-like behaviors were measured with the light-dark box test. Fecal corticosterone, cecal microbiota (composition and organic acids), plasma gut permeability (lipopolysaccharide-binding protein, LBP) and plasma inflammation (12 cytokines) markers were measured. Atypical behaviors were observed in female rats following UCMS, but no depressive-like behaviors were observed. Circulating concentrations of cytokines granulocyte-macrophage colony-stimulating factor and cytokine-induced neutrophil chemoattractant 1 were higher in UCMS-exposed female rats; plasma LBP and cecal organic acid levels remained unchanged. Our results reflect a resilient and adaptive phenotype for female SD rats. The relative abundance of taxa from the Clostridiales order and Desulfovibrionaceae family did, however, correlate both positively and negatively with anxiety-like behaviors and plasma cytokine concentrations, regardless of UCMS exposure, supporting the brain-to-gut influence of mild anxiety with a microbiota profile that may involve inflammatory pathways.

Baseline stool toxin concentration is associated with risk of recurrence in children with Clostridioides difficile infection.

In adults with Clostridioides difficile infection (CDI), higher stool concentrations of toxins A and B are associated with severe baseline disease, CDI-attributable severe outcomes, and recurrence. We evaluated whether toxin concentration predicts these presentations in children with CDI. We conducted a prospective cohort study of inpatients aged 2-17 years with CDI who received treatment. Patients were followed for 40 days after diagnosis for severe outcomes (intensive care unit admission, colectomy, or death, categorized as CDI primarily attributable, CDI contributed, or CDI not contributing) and recurrence. Baseline stool toxin A and B concentrations were measured using ultrasensitive single-molecule array assay, and 12 plasma cytokines were measured when blood was available. We enrolled 187 pediatric patients (median age, 9.6 years). Patients with severe baseline disease by IDSA-SHEA criteria (n = 34) had nonsignificantly higher median stool toxin A+B concentration than those without severe disease (n = 122; 3,217.2 vs 473.3 pg/mL; P = .08). Median toxin A+B concentration was nonsignificantly higher in children with a primarily attributed severe outcome (n = 4) versus no severe outcome (n = 148; 19,472.6 vs 429.1 pg/mL; P = .301). Recurrence occurred in 17 (9.4%) of 180 patients. Baseline toxin A+B concentration was significantly higher in patients with versus without recurrence: 4,398.8 versus 280.8 pg/mL (P = .024). Plasma granulocyte colony-stimulating factor concentration was significantly higher in CDI patients versus non-CDI diarrhea controls: 165.5 versus 28.5 pg/mL (P < .001). Higher baseline stool toxin concentrations are present in children with CDI recurrence. Toxin quantification should be included in CDI treatment trials to evaluate its use in severity assessment and outcome prediction.

Histological and Biochemical Analysis after Posterior Mandibular Displacement in Rats.

The present study aimed to investigate any biochemical and histological changes of the rat condyle and mandible in animals that had sustained mandibular growth restriction. Seventy-two male Wistar rats were divided into two equal groups, experimental and control. Each group consisted of three equal subgroups. The animals were sacrificed 30, 60, and 90 days after the start of the experiment. Blood samples were collected from the eye, and the osteoprotegerin (OPG), Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), and Macrophage Colony-Stimulating factor (MCSF)concentrations were measured by using enzyme-linked immunosorbent assay (ELISA) kits. A histological analysis was performed on the mandibular condyles. The blood serum values of OPG, RANKL, and MCSF did not exhibit any statistically significant difference between groups or subgroups. However, significant histological changes became evident after a histomorphometric condylar examination was performed. The Bone Surface/Total Surface ratio appeared reduced in the anterior and posterior regions of the condyle. In addition, the Posterior Condylar Cartilage Thickness was measured and determined to be significantly diminished. The present intervention that employed orthodontic/orthopedic devices did not prove to have any significant effect on the circulating proteins under study. Posterior displacement of the mandible may culminate only in local histological alterations in condylar cartilage thickness and its osseous microarchitecture.

Massive release of TH2 cytokines induced a cytokine storm during a severe mast cell activation event in a patient with indolent systemic mastocytosis

In subjects with systemic mastocytosis, the number of mast cells is elevated many fold. These patients frequently experience unpredictable and recurrent life-threatening mast cell activation (MCA) events. Our aim was to analyze the derangements of chemokine and cytokine concentrations during severe MCA attacks. Samples from a patient with indolent systemic mastocytosis were used for this study. Atotal of 41 chemokines and cytokines were simultaneously measured in triplicate and at multiple time points during 2 severe and 2 moderate MCA events. These were compared to 3 to 5 baseline samples, taken when clinical symptoms were not present. During the severe MCA event, which required 2 days of treatment in the intensive care unit, peak chemokine (C-C motif) ligand 3, IL-1ra, IL-5, IL-6, IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor concentrations were statistically significantly elevated 29-, 99-, 44-, 280-, 93-, 7-, and 6-fold above baseline, respectively. Ahighly similar pattern was observed during the second severe MCA event. In the moderate MCA event with PCR-proven influenza Ainfection, the TH1-associated cytokines INF-α, INF-γ, and TNF-α were only statistically significantly elevated 5- to 7-fold above baseline. The correlation coefficients between highly elevated histamine and cytokine concentrations during the acute phase were >95%, indicating the same cellular origin, possibly activated mast cells. One of the severe MCA events led to life-threatening symptoms over several days. During this event, the massive release of TH2 cytokines induced a hyperinflammatory state, fulfilling published criteria for cytokine release syndrome. Administration of IL-6- and IL-5-inhibiting biologicals might significantly shorten the acute phase of severe MCA events, likely offering significant clinical benefits to mastocytosis patients.

Plasma concentrations of granulocyte colony-stimulating factor (G-CSF) in patients with substance use disorders and comorbid major depressive disorder

Granulocyte colony–stimulating factor (G-CSF) has raised much interest because of its role in cocaine addiction in preclinical models. We explored the plasma concentrations of G-CSF in patients diagnosed with substance use disorder (SUD) and highly comorbid psychiatric disorders. In particular, we investigated the association between G-CSF concentrations and comorbid major depressive disorder (MDD) in patients with cocaine and alcohol use disorders (CUD and AUD, respectively). Additionally, patients with MDD but not SUD were included in the study. Three hundred and eleven participants were enrolled in this exploratory study: 136 control subjects, 125 patients with SUD (SUD group) from outpatient treatment programs for cocaine (N = 60, cocaine subgroup) and alcohol (N = 65, alcohol subgroup), and 50 patients with MDD but not SUD (MDD group) from primary-care settings. Participants were assessed based on DSM-IV-TR criteria, and a blood sample was collected to examine the plasma concentrations of G-CSF. G-CSF concentrations were negatively correlated with age in the entire sample (r = − 0.233, p < 0.001) but not in the patients with MDD. G-CSF concentrations were lower in patients with SUD than in controls (p < 0.05), specifically in the cocaine subgroup (p < 0.05). Patients with SUD and comorbid MDD had lower G-CSF concentrations than patients with SUD but not comorbid MDD or controls (p < 0.05). In contrast, patients with MDD but not SUD showed no differences compared with their controls. The negative association between G-CSF concentrations and age in the sample was not observed in patients with MDD. G-CSF concentrations were decreased in patients with SUD and comorbid MDD but not in patients with MDD. Therefore, G-CSF may be useful to improve the stratification of patients with dual diagnosis seeking treatment. Further investigation is needed to explore the impact of sex and type of drug on the expression of G-CSF.

Genetic Validation of the Palmitoylation/Depalmitoylation Cycle As a Drug Target in NRAS Mutant Hematologic Malignancies In Vivo

The C-terminal hypervariable region (HVR) of Ras proteins specifies post-translational modifications required for their subcellular localization and biologic function. The HVR of all four Ras isoforms terminates with a CAAX motif that is prenylated by farnesyltransferase, producing a weak membrane binding affinity that is further stabilized by a second signal motif. For K-Ras4b, this signal is provided by a polybasic lysine domain that allows direct localization of the protein at the plasma membrane (PM). By contrast, H-Ras, N-Ras, and K-Ras4a PM localization requires palmitoylation at cysteine(s) adjacent to the CAAX motif and is maintained through a dynamic cycle regulated by depalmitoylation and repalmitoylation. Importantly, these differences raise the provocative possibility that inhibiting palmitoylation could selectively target NRAS mutant cancer cells without toxicity to normal cells that retain intact K-Ras4b function.Indeed, Nras oncogenes with a cysteine-to-serine second site amino acid substitution at the palmitoylation site (C181S) exhibited defective transforming activity in hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo (Cuiffo et al., Blood 2010; Xu et al., Blood 2012). However, these studies relied on retroviral promoters to express oncogenic N-RasG12D that resulted in supra-physiological protein levels in HSPCs. To overcome this limitation, we retargeted embryonic stem cells that were used to generate Lox-STOP-Lox (LSL) NrasG12D/+ knock in mice ( Haigis et al., Nat Genet 2008 ) to create a NrasG12D,C181S allele (hereafter called NrasC181S). We then performed intercrosses to generate Mx1-Cre, NrasC181S/C181S mice as well as cohorts of congenic Mx1-Cre, NrasWT/WT and Mx1-Cre, NrasG12D/G12D control animals. As expected (Li et al ., Blood 2011; Wang et al ., Cell Cycle 2011), Mx1-Cre, NrasG12D/G12D mice that were injected with poly(I:C) at weaning had a mean white blood cell (WBC) count of 58.3 x 103/µL at six months of age (Fig.1 A) and died from hematologic disease after 225 days (range 119-267 days, n= 14, Fig. 1B). By contrast, Mx1-Cre, NrasC181S/C181S mice had normal WBC counts at the same time point (mean: 11.34x103 WBC/µL) and remained well until 19-20 months of age (n=12). The difference in survival between these groups was highly significant (p=3x10-6 by Kaplan-Meier analysis). Detailed analysis of the hematopoietic compartment at six months of age (n=5 per genotype) revealed profound splenomegaly (mean weight: 1.272g) in NrasG12D/G12D mice, but normal spleen weights in the NrasWT/WT and NrasC181S/C181S animals (Fig. 1C). The expansion of granulocyte-monocyte progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and more primitive CD48+ Lin- c-Kit+ Sca1+ (LKS) cells previously observed in NrasG12D/G12D mice was also rescued by the C181S second site mutation. Furthermore, in vitro culture of HSPC-enriched bone marrow cells over a range of granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations revealed normal cytokine sensitivity of NrasC181S/C181S cells, while NrasG12D/G12D cells were characterized by cytokine-independent growth and GM-CSF hypersensitivity as previously described. Importantly, Ras-GTP levels were similarly elevated in bone marrow cells from both NrasG12D/G12D and NrasC181S/C181S mice compared to wild-type cells. This indicates that the C181S mutation abrogates the cytokine hypersensitivity and increased cell proliferation that results from oncogenic N-RasG12D expression without reverting the canonical biochemical consequence of the oncogenic G12D substitution. This observation, in turn, suggests that abnormal subcellular localization and trafficking of oncogenic N-Ras potently suppresses myeloid transformation. Taken together, these in vivo studies directly validate the palmitoylation/depalmitoylation cycle as a therapeutic target in hematologic malignancies and potentially in other cancers characterized by oncogenic NRAS mutations. [Display omitted] DisclosuresFirestone:Calico Life Sciences: Employment.

Cytokine Profiles of Non-Small Cell Lung Cancer Patients Treated with Concurrent Chemoradiotherapy with Regards to Radiation Pneumonitis Severity.

The immunologic aspects of radiation pneumonitis (RP) are unclear. We analyzed variations in cytokine profiles between patients with grade (Gr) 0–1 and Gr ≥ 2 RP. Fifteen patients undergoing concurrent chemoradiotherapy for non-small cell lung cancer were included. Blood samples of 9 patients with Gr 0–1 and 6 with Gr ≥ 2 RP were obtained from the Biobank. Cytokine levels were evaluated using an enzyme linked immunosorbent assay at before radiotherapy (RT) initiation, 1, 3, and 6 weeks post-RT initiation, and 1 month post-RT completion. Concentrations of granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, IL-10, IL-13, IL-17, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β were analyzed; none were related to the occurrence of Gr ≥ 2 RP at pre-RT initiation. At 3 weeks, relative changes in the G-CSF, IL-6, and IFN-γ levels differed significantly between the groups (p = 0.026, 0.05 and 0.026, respectively). One month post-RT completion, relative changes of IL-17 showed significant differences (p = 0.045); however, relative changes in TNF-α, IL-10, IL-13, and TGF-β, did not differ significantly. Evaluation of changes in IL-6, G-CSF, and IFN-γ at 3 weeks after RT initiation can identify patients pre-disposed to severe RP. The mechanism of variation in cytokine levels in relation to RP severity warrants further investigation.

PSI-4 Changes in serum cortisol concentrations and cytokine profiles of growing pigs subjected to low-intensity heat stress

Abstract This study was to determine the impact of low-intensity heat stress on inflammatory biomarkers in serum, ileum, jejunum and lung lavage samples. Pigs (n = 64; 24.6 ± 3.7 kg BW) were housed four per pen in thermoneutral conditions (CON; 23.9°C ± 2.5°C) or constant high ambient temperatures (HS; 28.7°C ± 1.3°C) for 16 d, followed by thermoneutral conditions (24.4°C ± 1.8°C) for both groups through d 21, and subsequent heat stress (28.0°C ± 3.0°C) for both groups through d 30. Respiration rate (P &amp;lt; 0.01) was greater in HS than CON pigs from d 2 through d 15 (mean: 82.54 breaths/min vs. 59.26 breaths/min). HS pigs had greater cortisol concentration (40.1 ng/mL vs. 27.3 ng/mL) than CON on d 2, but lower concentrations on d 22 (32.9 ng/mL vs. 48.9 ng/mL) and d 25 (25.4 ng/mL vs. 42.0 ng/mL; treatment x day: P &amp;lt; 0.001). HS pigs tended to have greater serum IL1β (190.1 vs. 143.4 pg/mL; P = 0.06) and greater serum IL12 concentrations (914.9 vs. 785.4 pg/mL; P &amp;lt; 0.01) than CON pigs. HS pigs tended to have greater ileal mucosa IL6 concentrations than CON pigs (50.4 vs. 30.9 pg/mL; P = 0.08). Jejunal mucosa IL1α concentrations tended (P = 0.10) to be greater for CON than HS pigs (13.5 vs. 9.0 pg/mL) and CON pigs had greater jejunal IL12 concentrations (18.1 vs. 11.8 pg/mL) than HS pigs (P = 0.02). Bronchioalveolar lavage from HS pigs tended (P = 0.10) to have a greater granulocyte-macrophage colony-stimulating factor concentration (6.9 vs. 5.3 pg/mL) than CON pigs and had greater IL4 (7.7 vs. 4.2 pg/mL) and IL12 concentrations (34.8 vs. 24.5 pg/mL) than CON pigs (P = 0.01). Increased inflammatory activity and endocrine stress response occurred during low-intensity heat stress, indicating it may be more detrimental than previously accepted.

Interleukin (IL)-6: A Friend or Foe of Pregnancy and Parturition? Evidence From Functional Studies in Fetal Membrane Cells.

ObjectiveProtection of the fetus within the amniotic sac is primarily attained by remodeling fetal membrane (amniochorion) cells through cyclic epithelial to mesenchymal and mesenchymal to epithelial (EMT and MET) transitions. Endocrine and paracrine factors regulate EMT and MET during pregnancy. At term, increased oxidative stress forces a terminal state of EMT and inflammation, predisposing to membrane weakening and rupture. IL-6 is a constitutively expressed cytokine during gestation, but it is elevated in term and preterm births. Therefore, we tested the hypothesis that IL-6 can determine the fate of amnion membrane cells and that pathologic levels of IL-6 can cause a terminal state of EMT and inflammation, leading to adverse pregnancy outcomes.MethodsPrimary amnion epithelial cells (AECs) were treated with recombinant IL-6 (330, 1,650, 3,330, and 16,000 pg/ml) for 48 h (N = 5). IL-6-induced cell senescence (aging), cell death (apoptosis and necrosis), and cell cycle changes were studied using flow cytometry. Cellular transitions were determined by immunocytochemistry and western blot analysis, while IL-6 signaling (activation of signaling kinases) was measured by immunoassay. Inflammatory marker matrix metalloproteinase (MMP9) and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations were measured using a Fluorokine E assay and ELISA, respectively. Amniotic membranes collected on gestational day (D) 12 and D18 from IL-6 knockout (KO) and control C57BL/6 mice (N = 3 each) were used to determine the impact of IL-6 on cell transitions. Fold changes were measured based on the mean of each group.ResultsIL-6 treatment of AECs at physiologic or pathologic doses increased JNK and p38MAPK activation; however, the activation of signals did not cause changes in AEC cell cycle, cellular senescence, apoptosis, necrosis, cellular transitions, or inflammation (MMP9 and GM-CSF) compared to control. EMT markers were higher on D18 compared to D12 regardless of IL-6 status in the mouse amniotic sac.ConclusionPhysiologic and pathologic concentrations of IL-6 did not cause amnion cell aging, cell death, cellular transitions, or inflammation. IL-6 may function to maintain cellular homeostasis throughout gestation in fetal membrane cells. Although IL-6 is a good biomarker for adverse pregnancies, it is not an indicator of an underlying pathological mechanism in membrane cells.

Dexmedetomidine inhibits LPS-induced proinflammatory responses via suppressing HIF1α-dependent glycolysis in macrophages.

Dexmedetomidine, a highly selective α2-adrenoceptor agonist, has been reported to exert an anti-inflammatory effect in several animal models, but the mechanism remains unclear. Previous studies have shown that hypoxia inducible factor 1α-induced glycolysis is essential for the activation of inflammatory macrophages. However, whether dexmedetomidine influences hypoxia inducible factor 1α-induced glycolysis and thus exerts an anti-inflammatory effect has been poorly investigated. This study aims to elucidate the anti-inflammatory mechanism of dexmedetomidine involving the hypoxia inducible factor 1α-dependent glycolytic pathway. We showed that dexmedetomidine could suppress lipopolysaccharide-induced inflammatory cytokine production; inhibit the extracellular acidification rate, glucose consumption and lactate production; and decrease the expression of glycolytic genes in macrophages. The enhancement of glycolysis by the granulocyte-macrophage colony-stimulating factor or higher concentration of glucose could reverse the anti-inflammatory effect of dexmedetomidine on lipopolysaccharide-treated macrophages. Moreover, dexmedetomidine significantly inhibited the upregulation of hypoxia inducible factor 1α at the mRNA and protein levels. Genetic inhibition of hypoxia inducible factor 1α expression could reverse the anti-inflammatory effect of dexmedetomidine. Taken together, our results indicate that dexmedetomidine attenuates lipopolysaccharide-induced proinflammatory responses partially by suppressing hypoxia inducible factor 1α-dependent glycolysis in macrophages.

Neutralizing Complement C5a Protects Mice with Pneumococcal Pulmonary Sepsis

Community-acquired pneumonia and associated sepsis cause high mortality despite antibiotic treatment. Uncontrolled inflammatory host responses contribute to the unfavorable outcome by driving lung and extrapulmonary organ failure. The complement fragment C5a holds significant proinflammatory functions and is associated with tissue damage in various inflammatory conditions. The authors hypothesized that C5a concentrations are increased in pneumonia and C5a neutralization promotes barrier stabilization in the lung and is protective in pneumococcal pulmonary sepsis. The authors investigated regulation of C5a in pneumonia in a prospective patient cohort and in experimental pneumonia. Two complementary models of murine pneumococcal pneumonia were applied. Female mice were treated with NOX-D19, a C5a-neutralizing L-RNA-aptamer. Lung, liver, and kidney injury and the inflammatory response were assessed by measuring pulmonary permeability (primary outcome), pulmonary and blood leukocytes, cytokine concentrations in lung and blood, and bacterial load in lung, spleen, and blood, and performing histologic analyses of tissue damage, apoptosis, and fibrin deposition (n = 5 to 13). In hospitalized patients with pneumonia (n = 395), higher serum C5a concentrations were observed compared to healthy subjects (n = 24; 6.3 nmol/l [3.9 to 10.0] vs. 4.5 nmol/l [3.8 to 6.6], median [25 to 75% interquartile range]; difference: 1.4 [95% CI, 0.1 to 2.9]; P = 0.029). Neutralization of C5a in mice resulted in lower pulmonary permeability in pneumococcal pneumonia (1.38 ± 0.89 vs. 3.29 ± 2.34, mean ± SD; difference: 1.90 [95% CI, 0.15 to 3.66]; P = 0.035; n = 10 or 11) or combined severe pneumonia and mechanical ventilation (2.56 ± 1.17 vs. 7.31 ± 5.22; difference: 4.76 [95% CI, 1.22 to 8.30]; P = 0.011; n = 9 or 10). Further, C5a neutralization led to lower blood granulocyte colony-stimulating factor concentrations and protected against sepsis-associated liver injury. Systemic C5a is elevated in pneumonia patients. Neutralizing C5a protected against lung and liver injury in pneumococcal pneumonia in mice. Early neutralization of C5a might be a promising adjunctive treatment strategy to improve outcome in community-acquired pneumonia.

Effects of maternal L-proline supplementation on inflammatory cytokines at the placenta and fetus interface of mice.

Dietary L-proline (proline) supplementation during gestation enhances fetal survival and placental development in mice. The objective of the present study was to test the hypothesis that this beneficial effect of proline was associated with alterations in inflammatory response at the placenta and fetus interface. Populations of immune cells present in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry analysis. The concentrations of immunoglobulins in plasma, and the concentrations of cytokines in plasma, uterus, placenta, and amniotic fluid were measured using a bead-based immunoassay. The data showed that proline supplementation led to higher (P < 0.05) populations of B lymphocytes (CD3-CD19+), natural killer (NK) cells (CD3-NK1.1+), and dendritic cells (DCs, CD11c+MHCII+) in peripheral blood, as compared with the controls. Conversely, mice fed a proline-supplemented diet had a lower population of neutrophils (CD11b+F4/80-). Further study showed that proline supplementation decreased (P < 0.05) the concentrations of (1) interleukin (IL)-23, IL-1α, and IL-6 in plasma; (2) IL-6 in the uterus; and (3) tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein (MCP)-1, and IL-17 in the placenta; and (4) interferon (IFN)-γ in amniotic fluid, compared with controls. Conversely, proline supplementation resulted in higher (P < 0.05) concentrations of (1) IL-10, IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in plasma; (2) IL-10and IL-1α in the uterus; and (3) IL-1α, IL-1β, IL-10, IL-27, and IFN-β in amniotic fluid, compared with controls. Moreover, concentrations of immunoglobulin (Ig) G2b and IgM were enhanced (P < 0.05) by proline administration. Taken together, our results reveal a regulatory effect of proline in the immunological response at the maternal-fetal interface, which is critical for embryonic development and fetal survival.

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.

Infection-associated Immune Perturbations Resolve 1 Year Following Treatment for Loa loa.

We have previously demonstrated that eosinophil-associated processes underlie some of the differences in clinical presentation among patients with Loa loa infection prior to therapy and that some posttreatment adverse events appear to be dependent on eosinophil activation. We first conducted a retrospective review of 204 patients (70 microfilaria [MF] positive/134 negative) with Loa loa both before and following definitive therapy. We then measured filarial-specific antibodies, eosinophil- and Th2-associated cytokines, and eosinophil granule proteins in their banked serum prior to and at 1 year following definitive treatment. We also evaluated the influence of pretreatment corticosteroids and/or apheresis in altering the efficacy of treatment. Patients without circulating microfilariae (MF negative) not only had a higher likelihood of peripheral eosinophilia and increased antifilarial antibody levels but also had significantly increased concentrations of granulocyte-macrophage colony-stimulating factor, interleukin (IL) 5, and IL-4 compared with MF-positive patients. However, these differences had all resolved by 1 year after treatment, when all parameters approached the levels seen in uninfected individuals. Neither pretreatment with corticosteroids nor apheresis reduced the efficacy of the diethylcarbamazine used to treat these subjects. Our results highlight that, by 1 year following treatment, infection-associated immunologic abnormalities had resolved in nearly all patients treated for loiasis, and pretreatment corticosteroids had no influence on the resolution of the immunologic perturbations nor on the efficacy of diethylcarbamazine as a curative agent in loiasis. NCT00001230.

Granulocyte-Macrophage Colony-Stimulating Factor and CAR-T Technology for Solid Tumors in Experiment

Background. Cytokines are considered as important factors that enhance the efficacy of CAR-T cell therapy. Besides, they are key elements of the pathogenesis of cytokine release syndrome and neurotoxicity in applying the CAR-T technology. However, cytokine effects in the context of CAR-T therapy have not yet been properly studied. Aim. To quantitatively assess cytokine secretion using multiplex assay with co-incubation of anti-CD19 CAR-T lymphocytes with epithelial HeLa and A431 cell lines expressing CD19 on their surface. Materials &amp; Methods. T-lymphocytes were transduced with the lentiviral vector containing anti-CD19-CAR gene. CAR expression was tested based on GFP reporter using flow cytometry. To confirm a specific CAR-T cell activation response to tumor antigen, the levels of interleukin-2, interferon-γ, and tumor necrosis factor-α were measured by means of immunoassay. Cytotoxic activity of CAR-T lymphocytes obtained was examined with their direct co-culturing with target cells. The levels of cytokines isolated prior to and after incubation of targets with CAR-T cells were compared using multiplex assay. Results. The level of some proinflammatory cytokines (interleukin-6, interleukin-1β, interferon-γ) (P &lt; 0.01) increased. The difference in the levels of anti-inflammatory cytokines (interleukin-4, interleukin-10) was inconsiderable, and in the HeLa cell line experiment it was insignificant (p &gt; 0.05). The concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) was many times higher after incubation with CAR-T lymphocytes (p &lt; 0.01). Conclusion. The trial revealed multiple enhancement of GM-CSF, one of the key elements of the pathogenesis of cy tokine release syndrome and CAR-T-associated neurotoxicity. The results of further studies of GM-CSF can contribute to improving the efficacy of CAR-T therapy with considerably lower toxicity.

Abstract 1325: TPX-0022, a polypharmacology inhibitor of MET/CSF1R/SRC inhibits tumor growth by promoting anti-tumor immune responses

Abstract In the tumor microenvironment, tumor associated macrophages (TAMs) support tumor growth by suppressing antitumor immune responses and promoting angiogenesis, which is associated with disease progression and poor clinical outcomes. In contrast to the classic phagocytic and cytotoxic pro-inflammatory M1 phenotype of macrophages engulfing and digesting pathogens, TAMs often adopt the anti-inflammatory and immune regulatory M2 phenotype in response to colony stimulating factor 1 (CSF1), which is produced by either tumor cells or stroma cells. Signaling through colony stimulating factor 1 receptor (CSF1R), a receptor tyrosine kinase expressed on the surface of mononuclear phagocytes, is involved in the recruitment of TAMs and has been associated with tumor progression and suppression of the immune response. Thus, CSF1R represents a key therapeutic target. TPX-0022, a type I kinase inhibitor with a novel macrocyclic structure, has been designed and optimized to inhibit MET/CSF1R/SRC with enzymatic kinase inhibition IC50s of 0.14, 0.76 and 0.12 nM, respectively. In a Ba/F3 ETV6-CSF1R cell model, TPX-0022 inhibited both autophosphorylation of CSF1R with an IC50 &amp;lt;3 nM and cell growth with an IC50 of 14 nM. In addition, TPX-0022 effectively inhibited the growth of Ba/F3 ETV6-CSF1R xenograft tumors in vivo. In the CSF1/CSF1R signaling-dependent M-NFS-60 model, TPX-0022 not only exhibited potency with an IC50 of 0.3 nM under baseline condition, but also potently inhibited the growth of M-NFS-60 cells with an IC50 of 11.6 nM in the presence of exogenous CSF1 at 1 ng/mL concentration, a condition mimicking typical in vivo conditions in the presence of advanced tumors. In contrast, in our study, the potency of the type II CSF1R inhibitor PLX-3397 demonstrated a strong dependency on the concentration of mouse CSF1, as the anti-proliferation IC50 shifted from &amp;lt;0.1 nM to 146.4 nM when CSF1 concentration changed from baseline to 1 ng/mL. Finally, in the MC38 syngeneic mouse model, TPX-0022 effectively reduced TAMs, altered the polarity of TAMs toward a more M1 phenotype, increased cytotoxic T cells and inhibited the growth of MC38 tumors. These preclinical results demonstrated a potent CSF1R inhibitory activity of TPX-0022 and the ability of TPX-0022 to inhibit tumor growth and promote a pro-inflammatory anti-tumor microenvironment. Citation Format: Wei Deng, Dayong Zhai, Evan Rogers, Xin Zhang, Dong Lee, Jane Ung, Han Zhang, Jing Liu, Yuelie Lu, John Huang, Armin Graber, Zach Zimmerman, John Lim, Jeffrey Whitten, J. Jean Cui. TPX-0022, a polypharmacology inhibitor of MET/CSF1R/SRC inhibits tumor growth by promoting anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1325.

Peripheral adaptive immunity of the triple transgenic mouse model of Alzheimer\u2019s disease

BackgroundImmunologic abnormalities have been described in peripheral blood and central nervous system of patients suffering from Alzheimer’s disease (AD), yet their role in the pathogenesis still remains poorly defined.Aim and methodsWe used the triple transgenic mouse model (3xTg-AD) to reproduce Aβ (amyloid plaques) and tau (neurofibrillary tangles) neuropathologies. We analyzed important features of the adaptive immune system in serum, primary (bone marrow) as well as secondary (spleen) lymphoid organs of 12-month-old 3xTg-AD mice using flow cytometry and ELISPOT. We further investigated serum cytokines of 9- and 13-month-old 3xTg-AD mice using multiplex ELISA. Results were compared to age-matched non-transgenic controls (NTg).ResultsIn the bone marrow of 12-month-old 3xTg-AD mice, we detected decreased proportions of short-term reconstituting hematopoietic stem cells (0.58-fold, P = 0.0116), while lymphocyte, granulocyte, and monocyte populations remained unchanged. Our results also point to increased activation of both B and T lymphocytes. Indeed, we report elevated levels of plasma cells in bone marrow (1.3-fold, P = 0.0405) along with a 5.4-fold rise in serum IgG concentration (P < 0.0001) in 3xTg-AD animals. Furthermore, higher levels of interleukin (IL)-2 were detected in serum of 9- and 13-month-old 3xTg-AD mice (P = 0.0018). Along with increased concentrations of IL-17 (P = 0.0115) and granulocyte-macrophage colony-stimulating factor (P = 0.0085), these data support helper T lymphocyte activation with Th17 polarization.ConclusionCollectively, these results suggest that the 3xTg-AD model mimics modifications of the adaptive immunity changes previously observed in human AD patients and underscore the activation of both valuable and harmful pathways of immunity in AD.