ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

The high FKBP1A expression in WBCs as a potential screening biomarker for pancreatic cancer.

Given the limitation of current routine approaches for pancreatic cancer screening and detection, the mortality rate of pancreatic cancer cases is still critical. The development of blood-based molecular biomarkers for pancreatic cancer screening and early detection which provide less-invasive, high-sensitivity, and cost-effective, is urgently needed. The goal of this study is to identify and validate the potential molecular biomarkers in white blood cells (WBCs) of pancreatic cancer patients. Gene expression profiles of pancreatic cancer patients from NCBI GEO database were analyzed by CU-DREAM. Then, mRNA expression levels of three candidate genes were determined by quantitative RT-PCR in WBCs of pancreatic cancer patients (N = 27) and healthy controls (N = 51). ROC analysis was performed to assess the performance of each candidate gene. A total of 29 upregulated genes were identified and three selected genes were performed gene expression analysis. Our results revealed high mRNA expression levels in WBCs of pancreatic cancer patients in all selected genes, including FKBP1A (p < 0.0001), PLD1 (p < 0.0001), and PSMA4 (p = 0.0002). Among candidate genes, FKBP1A mRNA expression level was remarkably increased in the pancreatic cancer samples and also in the early stage (p < 0.0001). Moreover, FKBP1A showed the greatest performance to discriminate patients with pancreatic cancer from healthy individuals than other genes with the 88.9% sensitivity, 84.3% specificity, and 90.1% accuracy. Our findings demonstrated that the alteration of FKBP1A gene in WBCs serves as a novel valuable biomarker for patients with pancreatic cancer. Detection of FKBP1A mRNA expression level in circulating WBCs, providing high-sensitive, less-invasive, and cost-effective, is simple and feasible for routine clinical setting that can be applied for pancreatic cancer screening and early detection.

Analysis of Clinical, Immunological and Molecular Features of Leukocyte Adhesion Deficiency Type I in Egyptian Children.

PURPOSE: Leukocyte adhesion deficiency (LAD) represents a rare group of inherited inborn errors of immunity (IEI) characterized by bacterial infections, delayed umbilical stump separation, and autoimmunity. This single-center study aimed at describing the clinical, immunological, and molecular characterizations of 34 LAD-I Egyptian pediatric patients. METHODS: Details of 34 patients' personal medical history, clinical and laboratory findings were recorded; Genetic material from 28 patients was studied. Mutational analysis was done by Sanger sequencing. RESULTS: Omphalitis, skin and soft tissue infections with poorly healing ulcers, delayed falling of the umbilical stump, and recurrent or un-resolving pneumonia were the most common presentations, followed by chronic otitis media, enteropathy, periodontitis; and recurrent oral thrush. Persistent leukocytosis and neutrophilia were reported in all patients, as well as CD18 and CD11b deficiency. CD18 expression was < 2% in around 90% of patients. Sixteen different pathological gene variants were detected in 28 patients who underwent ITGß2 gene sequencing, of those, ten were novel and six were previously reported. Three families received a prenatal diagnosis. Patients were on antimicrobials according to culture's results whenever available, and on prophylactic Trimethoprim-Sulfamethoxazole 5 mg/kg once daily, with regular clinical follow up. Hematopoietic stem cell transplantation (HSCT) was offered for 4 patients. However due to severity of the disease and delay in diagnosis, 58% of the patients passed away in the first 2 years of life. CONCLUSION: This study highlights the importance of early diagnosis and distribution of ITGß2 gene mutation in Egyptian children. Further molecular studies, however, remain a challenging necessity for better disease characterization in the region.

Isolation and high-dimensional flow cytometric analysis of tumor-infiltrating leukocytes in a mouse model of colorectal cancer.

Colorectal cancer (CRC) is a complex and heterogeneous disease characterized by dysregulated interactions between tumor cells and the immune system. The tumor microenvironment plays a pivotal role in cancer initiation as well as progression, with myeloid immune cells such as dendritic cell and macrophage subsets playing diverse roles in cancer immunity. On one hand, they exert anti-tumor effects, but they can also contribute to tumor growth. The AOM/DSS colitis-associated cancer mouse model has emerged as a valuable tool to investigate inflammation-driven CRC. To understand the role of different leukocyte populations in tumor development, the preparation of single cell suspensions from tumors has become standard procedure for many types of cancer in recent years. However, in the case of AOM/DSS-induced colorectal tumors, this is still challenging and rarely described. For one, to be able to properly distinguish tumor-associated immune cells, separate processing of cancerous and surrounding colon tissue is essential. In addition, cell yield, due to the low tumor mass, viability, as well as preservation of cell surface epitopes are important for successful flow cytometric profiling of tumor-infiltrating leukocytes. Here we present a fast, simple, and economical step-by-step protocol for isolating colorectal tumor-associated leukocytes from AOM/DSS-treated mice. Furthermore, we demonstrate the feasibility of this protocol for high-dimensional flow cytometric identification of the different tumor-infiltrating leukocyte populations, with a specific focus on myeloid cell subsets.

The level of active DNA demethylation compounds in leukocytes and urine samples as potential epigenetic biomarkers in breast cancer patients.

The active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation results in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. Moreover, TETs' activity may be upregulated by ascorbate. Given that aberrant DNA methylation of genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in the active DNA demethylation process. The study included blood samples from breast cancer patients (n = 74) and healthy subjects (n = 71). We analyzed the expression of genes involved in the active demethylation process (qRT-PCR), and 5-mC and its derivatives level (2D-UPLC MS/MS). The ascorbate level was determined using UPLC-MS. Breast cancer patients had significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3 was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, the ascorbate level in the plasma of breast cancer patients was decreased with the accompanying increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2). The presented study indicates the role of TET3 in DNA demethylation in breast carcinogenesis.

The melano-macrophage: The black leukocyte of fish immunity.

Melanin and the process of melanin synthesis or melanogenesis have central roles in the immune system of insects, and production of melanin-synthesizing enzymes from their haemocytes may be induced following activation through danger signals. Melanin-containing macrophage-like cells have been extensively studied in amphibians and they are also present in reptiles. In fish, melano-macrophages are especially recognized with respect to melano-macrophage centres (MMCs), hypothesized to be analogues of germinal centres in secondary lymphoid organs of mammals and some birds. Melano-macrophages are in addition present in several inflammatory conditions, in particular melanised focal changes, or black spots, in the musculature of farmed Atlantic salmon, Salmo salar. Melanins are complex compounds that may be divided into different forms which all have the ability to absorb and scatter light. Other functions include the quenching of free radicals and a direct effect on the immune system. According to the common view held in the pigment cell community, vertebrate melanin synthesis with melanosome formation may only occur in cells of ectodermal origin. However, abundant information suggests that also myeloid cells of ectothermic vertebrates may be classified as melanocytes. Here, we discuss these opposing views and review relevant literature. Finally, we review the current status on the research concerning melanised focal muscle changes that represent the most severe quality problem in Norwegian salmon production, but also other diseases where melano-macrophages play important roles.

Integrins in Health and Disease-Suitable Targets for Treatment?

Integrin receptors are heterodimeric surface receptors that play multiple roles regarding cell-cell communication, signaling, and migration. The four members of the ß2 integrin subfamily are composed of an alternative α (CD11a-d) subunit, which determines the specific receptor properties, and a constant ß (CD18) subunit. This review aims to present insight into the multiple immunological roles of integrin receptors, with a focus on ß2 integrins that are specifically expressed by leukocytes. The pathophysiological role of ß2 integrins is confirmed by the drastic phenotype of patients suffering from leukocyte adhesion deficiencies, most often resulting in severe recurrent infections and, at the same time, a predisposition for autoimmune diseases. So far, studies on the role of ß2 integrins in vivo employed mice with a constitutive knockout of all ß2 integrins or either family member, respectively, which complicated the differentiation between the direct and indirect effects of ß2 integrin deficiency for distinct cell types. The recent generation and characterization of transgenic mice with a cell-type-specific knockdown of ß2 integrins by our group has enabled the dissection of cell-specific roles of ß2 integrins. Further, integrin receptors have been recognized as target receptors for the treatment of inflammatory diseases as well as tumor therapy. However, whereas both agonistic and antagonistic agents yielded beneficial effects in animal models, the success of clinical trials was limited in most cases and was associated with unwanted side effects. This unfavorable outcome is most probably related to the systemic effects of the used compounds on all leukocytes, thereby emphasizing the need to develop formulations that target distinct types of leukocytes to modulate ß2 integrin activity for therapeutic applications.

The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

OBJECTIVES: Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR). METHODS: MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). RESULTS: While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups. CONCLUSION: Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR.

CsIL-20, a tongue sole interleukin-20, negatively mediates leucocyte activity and antibacterial defense.

Interleukin-20 (IL-20), as an essential member of IL-10 family, plays vital roles in mammalian immunological response such as antimicrobial, inflammation, hematopoiesis, and immune diseases. In teleost, the study about immune antimicrobial function of IL-20 is largely scarce. In this article, we revealed the expression profiles and the immunological functions of the IL-20 (CsIL-20) in tongue sole Cynoglossus semilaevis. CsIL-20 is composed of 183 amino acid residues, with seven cysteine residues and a typical IL-10 domain which comprises six α-helices and two ß-sheets, and shares 34.4-71.2 % identities with other teleost IL-20. CsIL-20 was constitutively expressed in a variety of tissues and regulated by bacterial invasion, and the recombinant CsIL-20 (rCsIL-20) could bind to different bacteria. In vitro rCsIL-20 could interact with the membrane of peripheral blood leukocytes (PBLs), leading to the attenuation of reactive oxygen species (ROS) production and acid phosphatase activity in PBLs. In line with In vitro results, In vivo rCsIL-20 could obviously suppressed the host immune against bacterial infection. Furthermore, knockdown of CsIL-20 in vivo could markedly enhance the host antibacterial immunity. Collectively, these observations offer new insights into the negative effect of CsIL-20 on antibacterial immunity.

BNIP3 and DAPK1 methylation in peripheral blood leucocytes are noninvasive biomarkers for gastric cancer.

OBJECTIVE: The objective of this study is to comprehensively investigate the potential value of BNIP3 and DAPK1 methylation in peripheral blood leukocytes as a non-invasive biomarker for the detection of gastric cancer (GC), prediction of chemotherapy efficacy, and prognosis assessment. PATIENTS AND METHODS: Initially, multiple bioinformatic analyses were employed to explore the genetic landscape and biological effects of BNIP3 and DAPK1 in GC tissues. Subsequently, case-control and prospective follow-up studies were conducted to compare the differences in BNIP3 and DAPK1 methylation levels in peripheral blood leukocytes among GC patients and healthy controls, as well as between patients exhibiting sensitivity and resistance to platinum plus fluorouracil treatment, and between patients with varying survival outcomes of GC. Additionally, several predictive nomograms were constructed based on the identified CpG sites and relevant clinical parameters to forecast the occurrence of GC, chemotherapy efficacy, and prognosis. RESULTS: The upregulation of BNIP3 and DAPK1 was found to be associated with the development and poorer survival outcomes of GC. Furthermore, the expression of BNIP3/DAPK1 exhibited an inverse relationship with their DNA methylation levels and demonstrated a positive correlation with immune cell infiltration, as well as the IC50 values of 5-Fluorouracil and Cisplatin in GC tissues. Increased infiltration of macrophages in the high-expression groups was observed to be linked to unfavorable GC survival. In the case-control and follow-up studies, lower methylation levels of BNIP3 and DAPK1 were identified in the peripheral leukocytes of GC patients compared to healthy controls. Hypomethylation was also associated with more aggressive subtypes, diminished chemotherapy efficacy, and poorer survival outcomes in GC. CONCLUSION: The DNA methylation of BNIP3 and DAPK1 in peripheral blood leukocytes holds promise as a novel non-invasive biomarker for predicting the occurrence of GC, chemotherapy efficacy, and prognosis assessment.

Correlation of Short Leukocyte Telomeres and Oxidative Stress with the Presence and Severity of Lung Cancer Explored by Principal Component Analysis.

Lung cancer (LC) is the second most common malignancy and leading cause of cancer death. The potential "culprit" for local and systemic telomere shortening in LC patients is oxidative stress. We investigated the correlation between the peripheral blood leukocyte (PBL) telomere length (TL) and the presence/severity of LC and oxidative stress, and its usefulness as LC diagnostic marker. PBL TL was measured in 89 LC patients and 83 healthy subjects using the modified Cawthon RTq-PCR method. The relative PBL TL, found to be a potential diagnostic marker for LC with very good accuracy (P < 0.001), was significantly shorter in patients compared to the control group (CG) (P < 0.001). Significantly shorter telomeres were found in patients with LC TNM stage IV than in patients with stages I-III (P = 0.014), in patients without therapy compared to those on therapy (P = 0.008), and in patients with partial response and stable/progressive disease compared to those with complete response (P = 0.039). The total oxidant status (TOS), advanced oxidation protein products (AOPP), prooxidant-antioxidant balance (PAB) and C-reactive protein (CRP) were significantly higher in patients compared to CG (P < 0.001) and correlated negatively with TL in both patients and CG (P < 0.001). PCA showed a relation between PAB and TL, and between the EGFR status and TL. Oxidative stress and PBL telomere shortening are probably associated with LC development and progression.

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

Tumor necrosis factor-alpha mediates the negative association between telomere length and kidney dysfunction.

Aim/hypothesis: The relationship between peripheral blood leukocyte telomere length (LTL) and kidney dysfunction, especially in people with hypertension, remains unclear. No clinical study has explored the role of inflammation and oxidative stress in the relationship between LTL and kidney dysfunction. Therefore, we examined the relationship between baseline LTL and albuminuria progression and/or rapid renal function decline in Chinese patients with or without hypertension and investigated whether inflammation and oxidative stress played a mediating role in this relationship. Methods: We conducted a prospective study including 262 patients in a 7-year follow-up period from 2014 to 2021. Data on LTL, inflammation, oxidative markers, renal function, and urine protein levels were assessed. Kidney dysfunction was defined as either albuminuria progression, rapid decline in renal function, or the composite endpoint (albuminuria progression and rapid decline in renal function). Logistic regression and simple mediation models were used for the analysis. Results: In this cohort (mean age, 54.3±9.7 years; follow-up period, 5.9±1.1 years), 42(16.0%), 21(8.0%), and 59(22.5%) patients developed albuminuria progression, rapid eGFR decline, and the composite endpoint of kidney dysfunction, respectively. Logistic regression analysis showed that each standard deviation decrease of baseline LTL and the lower quartile (Q) of baseline LTL were significantly correlated with an increased risk of rapid decline in renal function (OR=1.83 [95% CI 1.07, 3.27] per 1SD, P=0.03; OR=7.57 [95% CI 1.25, 145.88] for Q1 vs. Q4, P for trend=0.031); and the composite endpoint of kidney dysfunction (OR=1.37 [95% CI 0.97, 1.96] per 1SD, borderline positive P=0.072; OR=2.96[95% CI 1.15, 8.2] for Q1 vs. Q4, P for trend=0.036). The mediating analysis showed that tumor necrosis factor (TNF)-a partly mediated the relationship between LTL and rapid decline in renal function (direct effect: ß=0.046 95%CI [0.006, 0.090],P=0.02; indirect effect: ß=0.013 95%CI [0.003, 0.020]), and the mediating proportion was 22.4%.In subgroup analyses, LTL was inversely associated with rapid decline in renal function or the composite endpoint of kidney dysfunction only in patients with hypertension (OR=49.07[3.72,211.12] vs.1.32[0.69,2.58] per 1SD, P for interaction=0.045;OR=3.10 [1.48, 7.52] vs.1.08[0.92,1.63] per 1SD, P for interaction=0.036). Conclusion: Baseline LTL could independently predict kidney dysfunction at follow-up, especially in participants with hypertension. TNF-a partially mediated the negative association between LTL and kidney dysfunction.

Post-operative C-reactive protein and white blood cells changes pattern following spinal deformity surgery and its clinical correlation.

OBJECTIVE: Following the changing pattern of post-operative CRP and WBC counts and compare them to the patient's clinical condition to find which one is better for early detection of early infection. METHODS: A total of 50 patients who underwent spinal deformity correction surgery without clinical signs of infection such as fever, wound redness, or discharge were enrolled in this prospective study. The C-reactive protein (CRP) and white blood cell (WBC) counts were measured the 2nd and 7th post-operative days. We try to detect the type of correlation between both CRP and WBCs level and clinical condition of patient regarding wound local condition. RESULTS: All cases showed high CRP by the 2nd day post-operative which decreased significantly but not reaching normal levels even by the 7th day. All cases showed elevated WBCs count by the 2nd day which decreased to normal levels by the 3rd day in 86% of patients, and by the 7th day, 94% of cases showed normal levels. In addition, WBCs in the 2nd day post-operative significantly positive correlated with fusion level and operative time. There was no significant correlation between WBCs and blood transfusion or age. No significant correlation between CRP and number of fusion levels, blood transfusion nor operative time. CONCLUSION: WBCs count returned earlier to normal levels than CRP in our cases, so monitoring early changes in the 1st week in WBCs count pattern is more indicative of an ongoing infectious process.

Reinforcement of colon anastomosis healing with leukocyte platelet-rich fibrin in rabbit model.

AIM: This study investigated the regenerative efficacy of leukocyte platelet-rich fibrin (L-PRF) on colon anastomotic healing in rabbits. MAIN METHODS: Thirty-six healthy male white New Zealand rabbits were subjected to complete transactions of the ascending colon. The rabbits were equally divided into two groups: the control group, where the transected colon ends were anastomosed by a simple interrupted suture pattern, and the L-PRF-treated group, in which L-PRF was wrapped entirely around the anastomotic line. The postoperative acute pain scale was assessed using the Bristol Rabbit Pain Scale before surgery and at each four-hour interval post-operatively. After euthanizing the rabbits, the adhesion degree score, anastomotic bursting pressure, and stenosis degree of the anastomotic colon were assessed, and histopathological examination at the 7th, 14th, and 28th days postoperatively. KEY FINDINGS: Rabbits in both groups showed a significant increase in pain scores compared to baseline. Postoperatively, the L-PRF group exhibited significantly lower pain scores, adhesion scores, and stenosis degrees than the control group. However, the anastomotic bursting pressure was significantly higher in the L-PRF group. Re-epithelialization, polymorphonuclear neutrophil infiltration, granulation tissue formation, and collagen deposition scores were improved considerably in the L-PRF group compared to the control group. Immunostaining of growth factor expression was significantly lower in the control than in the L-PRF group. SIGNIFICANCE: The L-PRF can augment collagen deposition, re-epithelialize the mucosa, promote angiogenesis, reduce adhesions, and diminish the stenosis degree scores. Therefore, it can be considered a promising aid in healing bowel anastomoses.

Endothelial AHR activity prevents lung barrier disruption in viral infection.

Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.

Predicted leukocyte telomere length and risk of myeloid neoplasms.

Maintenance of telomere length has long been established to play a role in the biology of cancer and several studies suggest that it may be especially important in myeloid malignancies. To overcome potential bias in confounding and reverse causation of observational studies, we use both a polygenic risk score (PRS) and inverse-variance weighted (IVW) Mendelian randomization (MR) analyses to estimate the relationship between genetically predicted leukocyte telomere length (LTL) and acute myeloid leukemia (AML) risk in 498 cases and 2099 controls and myelodysplastic syndrome (MDS) risk in 610 cases and 1759 controls. Genetic instruments derived from four recent studies explaining 1.23-4.57% of telomere variability were considered. We used multivariable logistic regression to estimate odds ratios (OR, 95% confidence intervals [CI]) as the measure of association between individual single-nucleotide polymorphisms and myeloid malignancies. We observed a significant association between a PRS of longer predicted LTL and AML using three genetic instruments (OR = 4.03 per ~1200 base pair [bp] increase in LTL, 95% CI: 1.65, 9.85 using Codd et al. [Codd, V., Nelson, C.P., Albrecht, E., Mangino, M., Deelen, J., Buxton, J.L., Hottenga, J.J., Fischer, K., Esko, T., Surakka, I. et al. (2013) Identification of seven loci affecting mean telomere length and their association with disease. Nat. Genet., 45, 422-427 427e421-422.], OR = 3.48 per one-standard deviation increase in LTL, 95% CI: 1.74, 6.97 using Li et al. [Li, C., Stoma, S., Lotta, L.A., Warner, S., Albrecht, E., Allione, A., Arp, P.P., Broer, L., Buxton, J.L., Alves, A.D.S.C. et al. (2020) Genome-wide association analysis in humans links nucleotide metabolism to leukocyte telomere length. Am. J. Hum. Genet., 106, 389-404.] and OR = 2.59 per 1000 bp increase in LTL, 95% CI: 1.03, 6.52 using Taub et al. [Taub, M.A., Conomos, M.P., Keener, R., Iyer, K.R., Weinstock, J.S., Yanek, L.R., Lane, J., Miller-Fleming, T.W., Brody, J.A., Raffield, L.M. et al. (2022) Genetic determinants of telomere length from 109,122 ancestrally diverse whole-genome sequences in TOPMed. Cell Genom., 2.] genetic instruments). MR analyses further indicated an association between LTL and AML risk (PIVW ≤ 0.049) but not MDS (all PIVW ≥ 0.076). Findings suggest variation in genes relevant to telomere function and maintenance may be important in the etiology of AML but not MDS.

CXCR4, regulated by HIF1A, promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation.

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45+ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.

Optimization of peritoneal fluid and leukocyte collection in patients with endometriosis.

OBJECTIVE: To propose a standardized protocol for peritoneal free fluid and leukocyte sample collection in women with endometriosis suitable for biomedical research on the basis of the surgical procedure, the clinical and technical conditions, and the quality of the samples obtained. DESIGN: Video showing the step-by-step collection procedure and the suitability of samples obtained for biomedical research. SUBJECTS: This study included 103 women with confirmed endometriosis by pathology analysis, who signed informed consent and were recruited from the Hospital "Virgen de la Arrixaca", Murcia, Spain. The study was approved by the Ethics Committee of University of Murcia (CEI 3156/2020). MAIN OUTCOME MEASURES: We analyzed the presence of free fluid in the peritoneal cavity and its relationship with hormonal treatment intake. In addition, the presence of blood contamination, the number of viable leukocytes and macrophages in free peritoneal fluid and lavages as well as their relationship with the lavage volume used, the body mass index, and the age of patients were analyzed. RESULTS: The presence of free peritoneal fluid, in which cells and molecules could be quantified, was scarce in the patients (21%), and it was not significantly related to hormonal treatment intake. The cell viability was higher than 98% in all collected samples; although 54% showed good quality and enough cellularity to be used in biomedical research, 40% were contaminated with blood and 6% had low cellularity. The number of leukocytes and macrophages recovered from the peritoneal lavages correlated positively with the lavage volume used and negatively with the body mass index and was independent of the age of the patients. CONCLUSION: We describe a standardized step-by-step procedure for peritoneal fluid and leukocyte collection in women with endometriosis, suitable for biomedical research, taking into account that not all women present free fluid in the peritoneal cavity. We propose to increase the lavage volume recommended by the World Endometriosis Research Foundation from 10 mL to at least 40 mL of sterile saline solution and its mobilization for at least 30 seconds within the peritoneal cavity, especially in patients with higher body mass index, to improve the efficiency of the procedure.

Targeted Nanocarriers Co-Opting Pulmonary Intravascular Leukocytes for Drug Delivery to the Injured Brain.

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.