Granulocyte Colony-stimulating Factor Concentrations Research Articles

Abstract 5329: Ovarian cancer predisposes neutrophils to form neutrophil extracellular traps(NETs)

Abstract Objective: Neutrophil extracellular trap (NETs) are neutrophil-derived extracellular DNA released in response to inflammation. In contrast to their primary host-defensive role, NETs have been recently reported to promote cancer progression. The aim of this study is to investigate the role of NETs in ovarian cancer progression. Method: The clinical data of ovarian cancer patients who underwent primary surgery at Osaka University Medical Hospital were retrospectively reviewed (n=340). The relationship between neutrophilia, peritoneal dissemination and prognosis were analyzed. Prior to initial treatment, peripheral blood was obtained from newly diagnosed ovarian cancer patients and examined for CBC and granulocyte-colony stimulating factor (G-CSF) concentration by ELISA. To evaluate NETs formation capacity, isolated neutrophils were stimulated with G-CSF and incubated in vitro to monitor NETs formation. The ascites was collected at the initial surgery and assessed for G-CSF concentration and for neutrophils population using flow cytometry. The omental tissues were pathologically examined for NETs using H&E-staining and immunostaining with anti-Citrullinated Histon3 (CitH3) antibody. To test NETs induction capacity, we treated human peripheral blood neutrophils with the conditioned media from 20 ovarian cancer cell lines and the serum from ovarian cancer patients. Then human ovarian cancer OVCAR8 cells were co-cultured with NETs to evaluate the effect of NETs on cancer cell adhesion. Results: Neutrophilia (neutrophils>7000/µl) was found in 39 patients (39/340,11.5%). Patients with neutrophilia were associated with advanced disease accompanied by peritoneal dissemination and compromised survival compared to patients without neutrophilia (5-year PFS 40.0 vs 59.9 %, p 0.0027). In these patients, serum G-CSF was upregulated. Neutrophils from ovarian cancer patients formed NETs significantly earlier than those from benign tumor patients. Ovarian cancer patients with neutrophilia showed elevated G-CSF concentration and increased neutrophils proportion in their ascites. Omental tissues from patients with neutrophilia showed NETs foci close to the disseminated lesions. G-CSF strongly induced neutrophils to form NETs in vitro, which was observed in some conditioned media from ovarian cancer cell lines and serum from ovarian cancer patients as well. The adhesion of ovarian cancer cell was increased by co-incubation with NETs. Conclusion: Neutrophilia is associated with peritoneal dissemination and poor prognosis in ovarian cancer. One of the underlying mechanism of neutrophilia is upregulated G-CSF, which stimulate neutrophils to form NETs. NETs may have a causative effect on ovarian cancer dissemination. Citation Format: Gaku Yamamoto, Mahiru Kawano, Michiko Bun, Koutaro Shimura, Aska Toda, Koji Nakamura, Yasuto Kinose, Michiko Kodama, Kae Hashimoto, Kenjiro Sawada, Tadashi Kimura. Ovarian cancer predisposes neutrophils to form neutrophil extracellular traps(NETs) [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 5329.

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.

Plasma cytokine concentrations of children with autism spectrum disorder and neurotypical siblings

Several studies of autism spectrum disorder (ASD) have shown cytokine dysregulation in children with ASD, leading to a consideration of the immune theory of the ASD etiopathogenesis and a debate about cytokines as potential biomarkers of ASD. However, the results of these studies are still inconsistent. Overall, studies comparing the cytokine levels of children with ASD and neurotypical siblings achieved relatively different results than studies with control groups of non-siblings. The studies suggest that the immune profile of siblings of individuals with ASD serving as control is more similar to children with ASD than the profile of non-siblings. However, there are still only a few studies with control groups including neurotypical siblings of children with ASD.The aim of our study was to determine whether the concentration of plasma cytokine levels may differentiate children with ASD from their neurotypical siblings. The sample consisted of 40 children with ASD (mean age 7.11 years, SD 2.9) and 21 neurotypical siblings (mean age 7.38, SD 3.3). Levels of 20 cytokines were included into the statistical analysis. A multiple logistic regression model using multiple corrections showed that an increase in log-transformed plasma G-CSF (granulocyte colony stimulating factor) concentration is associated with an increased risk of the child being diagnosed as an ASD case (OR = 4.35, 95% CI 1.77, 10.73). Although the significantly increased concentration of G-CSF suggests a slightly different activity of the immune system of children with ASD, the overall cytokine profile of their siblings appeared to be very similar.

Dynamic changes in radiological parameters, immune cells, selected miRNAs, and cytokine levels in peripheral blood of patients with severe COVID‑19.

The present study aimed to investigate the dynamic changes in peripheral blood leucocyte subpopulations, cytokine and miRNA levels, and changes in computed tomography (CT) scores in patients with severe coronavirus disease 2019 (COVID-19) (n=14) and age-matched non-COVID-19 volunteers (n=17), which were included as a reference control group. All data were collected on the day of patient admission (day 0) and on the 7th, 14th and 28th days of follow-up while CT of the lungs was performed on weeks 2, 8, 24 and 48. On day 0, lymphopenia and leucopenia were detected in most patients with COVID-19, as well as an increase in the percentage of banded neutrophils, B cells, and CD4+ Treg cells, and a decrease in the content of PD-1low T cells, classical, plasmacytoid, and regulatory dendritic cells. On day 7, the percentage of T and natural killer cells decreased with a concurrent increase in B cells, but returned to the initial level after treatment discharge. The content of different T and dendritic cell subsets among CD45+ cells increased during two weeks and remained elevated, suggesting the activation of an adaptive immune response. The increase of PD-1-positive subpopulations of T and non-T cells and regulatory CD4 T cells in patients with COVID-19 during the observation period suggests the development of an inflammation control mechanism. The levels of interferon γ-induced protein 10 (IP-10), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 decreased on day 7, but increased again on days 14 and 28. C-reactive protein and granulocyte colony-stimulating factor (G-CSF) levels decreased gradually throughout the observation period. The relative expression levels of microRNA (miR)-21-5p, miR-221-3p, miR-27a-3p, miR-146a-5p, miR-133a-3p, and miR-126-3p were significantly higher at the beginning of hospitalization compared to non-COVID-19 volunteers. The plasma levels of all miRs, except for miR-126-3p, normalized within one week of treatment. At week 48, CT scores were most prominently correlated with the content of lymphocytes, senescent memory T cells, CD127+ T cells and CD57+ T cells, and increased concentrations of G-CSF, IP-10, and macrophage inflammatory protein-1α.

Granulocyte-colony stimulating factor does not prevent in vitro cisplatin-induced germ cell reduction in immature human and mouse testis

BackgroundCurrently there are no established fertility preservation options for pre-pubertal boys facing cancer treatment. Granulocyte-colony stimulating factor (G-CSF) treatment has been proposed to be chemoprotective against spermatogonial cell loss in an alkylating chemotherapy model of busulfan treated adult mice. Having previously shown that exposure to the alkylating-like chemotherapy cisplatin resulted in a reduction in germ cell numbers in immature human testicular tissues, we here investigate whether G-CSF would prevent cisplatin-induced germ cell loss in immature human and mouse (fetal and pre-pubertal) testicular tissues.MethodsOrganotypic in vitro culture systems were utilised to determine the effects of clinically-relevant concentrations of G-CSF in cisplatin-exposed immature testicular tissues. Human fetal (n = 14 fetuses) and mouse pre-pubertal (n = 4 litters) testicular tissue pieces were cultured and exposed to cisplatin or vehicle control for 24 hrs and analysed at 72 and 240 hrs post-exposure. Combined G-CSF and cisplatin exposure groups explored varying concentrations and duration of G-CSF supplementation to the culture medium (including groups receiving G-CSF before, during and after cisplatin exposure). In addition, effects of G-CSF supplementation alone were investigated. Survival of total germ cell and sub-populations were identified by expression of AP2γ and MAGE-A4 for human gonocytes and (pre)spermatogonia, respectively, and MVH and PLZF, for mouse germ cells and putative spermatogonial stem cells (SSCs) respectively, were quantified.ResultsExposure to cisplatin resulted in a reduced germ cell number in human fetal and mouse pre-pubertal testicular tissues at 240 hrs post-exposure. Germ cell number was not preserved by combined exposure with G-CSF using any of the exposure regimens (prior to, during or after cisplatin exposure). Continuous supplementation with G-CSF alone for 14 days did not change the germ cell composition in either human or mouse immature testicular tissues.ConclusionsThis study demonstrates that exposure to G-CSF does not prevent cisplatin-induced germ cell loss in immature human and mouse testicular tissues in an in vitro system.

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.

Granulocyte colony-stimulating factor in bronchoalveolar lavage fluid is a potential biomarker for prognostic prediction of idiopathic pulmonary fibrosis.

Background/AimsNeutrophilia is frequently observed in bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) patients. Granulocyte colony-stimulating factor (G-CSF) is a potent neutrophil-activating glycoprotein. However, the clinical implications of G-CSF remain poorly understood.in patients with IPF. Therefore, we evaluated the relationship between the G-CSF concentration in BALF and the progression of fibrosis, including in terms of the decline in lung function and long-term survival rate.MethodsG-CSF concentrations were measured in BALF using enzyme-linked immunosorbent assay (ELISA). The survival rate was estimated using Kaplan-Meier survival analyses.ResultsG-CSF protein levels were significantly higher in IPF (n = 87; 1.88 [0 to 5.68 pg/mL]), nonspecific interstitial pneumonia (n = 22; 0.58 [0 to 11.64 pg/mL]), and hypersensitivity pneumonitis (n = 19; 2.48 [0.46 to 5.71 pg/mL]) patients than in normal controls (n = 33; 0 [0 to 0.68 pg/mL]) (all p < 0.01). A receiver operating characteristic curve showed a difference in G-CSF levels between IPF and NC (area under the curve, 0.769): The G-CSF cut-off of 0.96 pg/mL indicated 84.9% specificity and 63.2% sensitivity for IPF. The survival rate was significantly lower in the group with G-CSF > 2.872 pg/mL than in the group with ≤ 2.872 pg/mL (hazard ratio, 2.69; p = 0.041). The annual decline in diffusing capacity of the lung for carbon monoxide was positively correlated with the G-CSF level (p = 0.018).ConclusionsG-CSF may participate in the development of IPF and be useful for predicting the prognosis of IPF. Therefore, G-CSF should be analyzed in BALF, in addition to differential cell counts.

Granulocyte-colony stimulating factor: Missing link for stratification of type 2–high and type 2–low chronic rhinosinusitis patients

Chronic rhinosinusitis (CRS) is a heterogeneous disease, with patients having either a high or low type 2 inflammatory endotype. Whereas the type 2-high group is well characterized by IL-5 expression, the type 2-low group, consisting of approximately 20% of CRS with and 50% of CRS without nasal polyp patients, lacks a clear biomarker profile and thus specific therapeutic targets. The aim was to identify underlying molecular pathways of type 2-low CRS, as stratification of patients may allow improvement of personalized treatments. Luminex assays were performed to analyze proteins in nasal secretions and tissues of CRS patients. Immunostainings were analyzed for differences in neutrophils, granulocyte-colony stimulating factor (G-CSF), and its receptor in nasal tissue. Neutrophils were isolated from blood of healthy volunteers and stimulated with G-CSF. Effects on apoptosis and neutrophil activity were analyzed with flow cytometry. G-CSF was significantly upregulated in nasal tissue and secretion fluid of type 2-low CRS patients compared to type 2-high patients. In nasal polyp tissue of type 2-low patients, a large infiltration of neutrophils expressing both G-CSF and its receptor was detected, suggesting the presence of a neutrophil-intrinsic autocrine survival mechanism. In response to G-CSF, neutrophils were in an activated state and were resistant to apoptosis, possibly contributing to a chronic inflammation. Of interest, type 2-high nasal polyp patients treated with IgE-blocking omalizumab had increased G-CSF concentrations compared to before treatment. G-CSF is an important cytokine regulating neutrophils in type 2-low CRS and has potential in the diagnosis and therapy of the disease.

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.

Dietary supplementation of a Saccharomyces cerevisiae fermentation product attenuates exercise-induced stress markers in young horses.

Mitigation of exercise-induced stress is of key interest in determining ways to optimize performance horse health. To test the hypothesis that dietary supplementation of a Saccharomyces cerevisiae fermentation product would decrease markers of exercise-induced stress and inflammation in young horses, Quarter Horse yearlings (mean ± SD; 9 ± 1 mo) were randomly assigned to receive either no supplementation (CON; n = 8) or 21 g/d S. cerevisiae fermentation product (10.5 g/feeding twice daily; SCFP; n = 10) top-dressed on a basal diet of custom-formulated grain as well as ad libitum Coastal bermudagrass hay. After 8 wk of dietary treatments, horses underwent a 2-h submaximal exercise test (SET) on a free-stall mechanical exerciser. Serum was collected before dietary treatment supplementation (week 0), at week 8 pre-SET, and 0, 1, and 6 h post-SET and analyzed for concentrations of cortisol and serum amyloid A (SAA) by commercial enzyme-linked immunosorbent assay (ELISA) and for cytokine concentrations by commercial bead-based ELISA. Data were analyzed using linear models with repeated measures in SAS v9.4. From week 0 to 8 (pre-SET), serum cortisol decreased (P = 0.01) and SAA did not change, but neither were affected by diet. Serum concentrations of all cytokines decreased from week 0 to 8 (P ≤ 0.008), but granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and interleukin-8 (IL-8) decreased to a greater extent in CON than in SCFP horses (P ≤0.003). In response to the week 8 SET, serum cortisol increased in all horses (P < 0.0001) but returned to pre-SET levels by 1 h post-SET in horses receiving SCFP. At 6 h post-SET, cortisol concentrations in CON horses returned to pre-SET concentrations, whereas cortisol declined further in SCFP horses to below pre-SET levels (P = 0.0002) and lower than CON (P = 0.003) at that time point. SAA increased at 6 h post-SET in CON (P < 0.0001) but was unchanged through 6 h in SCFP horses. All cytokines except G-CSF increased in response to the SET (P < 0.0001) but showed differing response patterns. Concentrations of IL-1β, IL-6, and tumor necrosis factor-alpha were lesser (P ≤ 0.05), and concentrations of G-CSF and IL-18 tended to be lesser (P ≤ 0.09) in SCFP compared with CON horses throughout recovery from the SET. In summary, 8 wk of dietary supplementation with 21 g/d of SCFP may mitigate cellular stress following a single, prolonged submaximal exercise bout in young horses.

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.

Granulocyte Colony-Stimulating Factor Worsens the Outcome of Experimental Klebsiella pneumoniae Pneumonia Through Direct Interaction With the Bacteria

AbstractBesides its well-established effects on granulocytopoiesis, granulocyte colony-stimulating factor (G-CSF) has been shown to have direct effects on the recruitment and bactericidal ability of neutrophils, resulting in improved survival of experimentally infected animals. We studied the effect of G-CSF on the course of experimental pneumonia induced by Klebsiella pneumoniae, an important gram-negative bacillary pulmonary pathogen. Using a highly reproducible murine model, we here show the paradoxical finding that mortality from infection was significantly increased when animals received G-CSF before induction of pneumonia. Administration of G-CSF promoted replication of bacteria in the liver and spleen, thus indicating an impairment rather than an enhancement of antibacterial mechanisms. By contrast, a monoclonal antibody against Klebsiella K2 capsule significantly reduced bacterial multiplication in the lung, liver, and spleen, and abrogated the increased mortality caused by G-CSF. In vitro studies showed a direct effect of G-CSF on K pneumoniaeresulting in increased capsular polysaccharide (CPS) production. When bacteria were coincubated with therapeutically achievable concentrations of G-CSF, phagocytic uptake and killing by neutrophils was impaired. Western blot analysis showed three binding sites of G-CSF to K pneumoniae. Binding of 125I-G-CSF to K pneumoniae was displaced by an excess of unlabeled G-CSF, whereas an unrelated cytokine, interleukin-1α, did not compete with G-CSF binding to the bacteria. Thus, in this model, the direct effect of G-CSF on a bacterial virulence factor, CPS production, outweighed any beneficial effect of G-CSF on recruitment and stimulation of leukocytes.

HLA-G LEVELS IN FOLLICULAR FLUID ARE NOT ASSOCIATED WITH FOLLICULAR G-CSF CONCENTRATIONS IN WOMEN UNDERGOING IN VITRO FERTILIZATION

Recently, soluble human leukocyte antigen-G (sHLA-G) and granulocyte colony-stimulating factor (G-CSF) quantified in the follicular fluid have been proposed as non-invasive biomarkers of oocyte competence in both stimulated and natural IVF/ICSI cycles. sHLA-G, G-CSF and their receptors were described in various human fetal tissues and it was suggested that they play an important role in ovulation, oocyte maturation, embryo development and implantation. However, the possible association between these two molecules in the follicular fluid has not been studied yet. The purpose of the present study was to determine whether the sHLA-G levels are associated with the G-CSF levels in follicular fluid of women undergoing in vitro fertilization. This is a cohort study of 56 women undergoing stimulated cycles and in vitro fertilization between September 2019 and November 2019 at Nadezhda Women’s Health Hospital. Follicular fluid from up to six clear follicular aspirates was collected during regular egg collection procedure of 56 patients. Samples were stored at -20°C until measurement. Follicular fluid levels of sHLA-G and G-CSF were evaluated by ELISA kits (Elabscience; E-EL-H1663 and RayBiotech; ELH-GCSF-1, respectively) according to the manufacturer’s instructions. The absorption was measured on a microplate reader (Beckman Coulter DTX 880 Multimode detector) at 450 nm. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). sHLA-G was detected in all 56 follicular fluid samples, with a median value of 47.52 IU/ml and a range of 13.85-63.06 IU/ml. Follicular fluid G-CSF was also detected in all samples and ranged between 3 and 116 pg/ml with a median of 16.23 pg/ml. Measured sHLA-G levels did not show a significant correlation with G-CSF concentration in follicular fluid (P = 0.63). This is the first report regarding the association between follicular fluid levels of sHLA-G and G-CSF. Although the increased secretion of both sHLA-G and G-CSF usually correlates with better oocyte quality and implantation success, our findings suggest that they are not related to each other.

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.

A paracrine interaction between granulosa cells and leukocytes in the preovulatory follicle causes the increase in follicular G-CSF levels

ObjectiveFollicular granulocyte colony-stimulating factor (G-CSF) is a new biomarker of oocyte quality and embryo implantation in in vitro fertilization (IVF) cycles. Its role in reproduction is poorly understood. Our study aimed to investigate the mechanisms and cells responsible for G-CSF production in the preovulatory follicle.DesignLaboratory research study.SettingSingle-center study.InterventionsGranulosa cells and leukocytes were isolated from the follicular fluids (FF) or the blood of women undergoing IVF and from the blood of a control group of women with spontaneous ovulatory cycles to perform cocultures.Main outcome measureG-CSF-secreted protein was quantified in the conditioned media of cocultures.ResultsG-CSF secretion was considerably increased in cocultures of granulosa cells and leukocytes. This effect was maximal when leukocytes were isolated from the blood of women in the late follicular phase of the menstrual cycle or from the FF of women undergoing IVF. The leukocyte population isolated from the FF samples of women undergoing IVF had a higher proportion of granulocytes than that isolated from the corresponding blood samples. Leukocytes induced the synthesis and secretion of G-CSF by granulosa cells. Among a range of other FF cytokines/chemokines, only growth-regulated oncogene alpha (GROα) was also increased.ConclusionThe notable rise in G-CSF at the time of ovulation coincides with the accumulation of follicular granulocytes, which stimulate G-CSF production by granulosa cells via paracrine interactions. High follicular G-CSF concentrations may occur in follicles with optimal granulosa–leukocyte interactions, which could explain the increased implantation rate of embryos arising from these follicles.

Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood.

Granulocyte colony‐stimulating factor (G‐CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G‐CSF, IL‐6, and MCP‐1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G‐CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G‐CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP‐mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G‐CSF. Although CoPP is used to activate Nrf2/HO‐1 axis, the observed effects are Nrf2/HO‐1 independent. Concluding, CoPP increases expression of mobilization‐related cytokines and has superior mobilizing efficiency compared with recombinant G‐CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.

Absence of correlation between follicular fluid volume and follicular granulocyte colony-stimulating factor, a predictor of embryo implantation and successful delivery

Follicular granulocyte colony-stimulating factor (G-CSF) is a documented marker of embryo implantation potential. The primary objective was to determine whether follicular G-CSF levels correlate with follicular fluid volume. The secondary objectives were to assess whether follicular G-CSF is associated with oocyte maturity at the time of harvest and with delivery rate after fresh or frozen embryo transfer. Thirty-two patients undergoing intracytoplasmic sperm injection (ICSI) cycles were recruited (Centre de Procréation Médicalement Assistée (CPMA), University of Liège, Belgium). A total of 211 follicular fluid (FF) samples were individually collected at the time of oocyte harvest. FF volume was recorded, and G-CSF concentration was assessed by ELISA. The embryos were individually cultured in vitro. Their implantation and live birth rates were recorded after fresh and frozen embryo transfers. The follicular fluid volume did not correlate with the follicular G-CSF concentration. There were no differences in follicular G-CSF levels between mature and immature oocytes. The probability of successful implantation and delivery was increased for embryos with FF containing a high G-CSF concentration. There was a trend toward lower follicular G-CSF levels in cases of miscarriage. Therefore, follicular fluid volume cannot be a substitute for follicular G-CSF as a marker of embryo implantation ability.

Clinical performance of a specific granulocyte colony stimulating factor ELISA to determine its concentration in follicular fluid as a predictor of implantation success during in vitro fertilization

This study aimed to demonstrate the clinical performance of an ultra-sensitive follicular fluid (FF) granulocyte colony stimulating factor (G-CSF) immunoassay to confirm previous work, indicating a correlation between FF G-CSF concentration and live birth potential of the corresponding embryo after in vitro fertilization. This study was a noninterventional, prospective, diagnostic clinical multicentric study conducted between August 2012 and January 2014 with 396 single embryo transfers (SETs) from 278 subjects. During oocyte retrieval, FF was individually collected. Embryo morphology and implantation success were evaluated. The implantation success rate in the high G-CSF group (32.3%) was higher than the overall rate (27.5%). Similarly, for embryos with optimal morphology, implantation success rates were highest among those in the high G-CSF concentration category (34.5%) compared with low (19.6%) and intermediate (29.8%) G-CSF concentration categories. Significant differences in mean G-CSF concentrations were observed between the study sites. To minimize bias, analyses were repeated using data from the center with the largest number of SETs. In alignment with the overall analysis, this center demonstrated a 43% greater probability of implantation for optimal embryos with high G-CSF compared to the general implantation rate among optimal embryos and a 327% increase compared with the implantation rate of optimal embryos with low G-CSF.Trial registration: ClinicalTrials.gov identifier: NCT02593513.Trial registration: ClinicalTrials.gov identifier: NCT02593461.Trial registration: ClinicalTrials.gov identifier: NCT02586272.

T helper type cytokines in sepsis: time-shared variance and correlation with organ dysfunction and hospital mortality

ObjectiveWe evaluated the kinetics of cytokines belonging to the T helper1 (Th1), Th2, and Th17 profiles in septic patients, and their correlations with organ dysfunction and hospital mortality. MethodsThis was a prospective observational study in a cohort of septic patients admitted to the intensive care units (ICU) of three Brazilian general hospitals. A total of 104 septic patients and 53 health volunteers (controls) were included. Plasma samples were collected within the first 48h of organ dysfunction or septic shock (0D), after seven (D7) and 14 days (D14) of follow-up. The following cytokines were measured by flow cytometry: Interleukin-1β (IL-1β), IL-2, IL-6, IL-8, IL-10, IL-12/23p40, IL-17, IL-21, tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF). ResultsIL-6, IL-8, G-CSF and IL-10 concentrations were higher in septic patients than in controls (p<0.001), while IL-12/23p40 presented higher levels in the controls (p=0.003). IL-6, IL-8 and IL-17 correlated with Sequential [Sepsis-related] Organ Failure Assessment (SOFA) D0, D1 and D3 (except for IL-6 at D0). IL-8 was associated with renal and cardiovascular dysfunction. In a mixed model analysis, IL-10 estimated means were lower in survivors than in deceased (p=0.014), while IL-21 had an estimated mean of 195.8pg/mL for survivors and 98.5 for deceased (p=0.03). Cytokines were grouped in four factors according to their kinetics over the three dosages (D0, D7, D14). Group 1 encompassed IL-6, IL-8, IL-10, IL-1β, and G-CSF while Group 3 encompassed IL-17 and IL-12/23p40. Both correlated with SOFA (D0) (p=0.039 and p=0.003, respectively). IL-21 (Group 4) was higher in those who survived. IL-2, TNF-α and GM-CSF (Group 2) showed no correlation with outcomes. ConclusionInflammatory and anti-inflammatory cytokines shared co-variance in septic patients and were related to organ dysfunctions and hospital mortality.

Chemokines and Growth Factors Produced by Lymphocytes in the Incompetent Great Saphenous Vein.

The role of cytokines in the pathogenesis of chronic venous disease (CVD) remains obscure. It has been postulated that oscillatory flow present in incompetent veins causes proinflammatory changes. Our earlier study confirmed this hypothesis. This study is aimed at assessing chemokines and growth factors (GFs) released by lymphocytes in patients with great saphenous vein (GSV) incompetence. In 34 patients exhibiting reflux in GSV, blood was derived from the cubital vein and from the incompetent saphenofemoral junction. In 12 healthy controls, blood was derived from the cubital vein. Lymphocyte culture with and without stimulation by phytohemagglutinin (PHA) was performed. Eotaxin, interleukin 8 (IL-8), macrophage inflammatory protein 1 A and 1B (MIP-1A and MIP-1B), interferon gamma-induced protein (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 5 (IL-5), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF) were assessed in culture supernatants by a Bio-Plex assay. Higher concentrations of eotaxin and G-CSF were revealed in the incompetent GSV, compared with the concentrations in the patients' upper limbs. The concentrations of MIP-1A and MIP-1B were higher in the CVD group while the concentration of VEGF was lower. In the stimulated cultures, the concentration of G-CSF proved higher in the incompetent GSV, as compared with the patients' upper limbs. Between the groups, the concentration of eotaxin was higher in the CVD group, while the IL-5 and MCP-1 concentrations were lower. IL-8, IP-10, FGF, GM-CSF, and PDGF-BB did not reveal any significant differences in concentrations between the samples. These observations suggest that the concentrations of chemokines and GFs are different in the blood of CVD patients. The oscillatory flow present in incompetent veins may play a role in these changes. However, the role of cytokines in CVD requires further study.