Active Bone Marrow Research Articles

Immature granulocytes: Innovative biomarker for SARS‑CoV‑2 infection.

Immature granulocytes (IGs) include metamyelocytes, myelocytes and promyelocytes, and are the precursors of neutrophils. Increased IG counts found in peripheral blood indicate an enhanced bone marrow activity. In addition, IGs have been evaluated in numerous clinical conditions, such as severe acute pancreatitis, systemic inflammatory response syndrome and infectious complications following open-heart surgery under cardiopulmonary bypass. Neutrophils are considered to play a crucial role in the host defense during bacterial and fungal infections, and are involved in the antiviral immune response. Numerous studies have reported the role of neutrophils in coronavirus disease 2019 (COVID-19) infection, concluding that the percentage of neutrophils may be a predictor of the severity of COVID-19 infection. There has been limited research regarding the role of neutrophil precursors in viral infections, including severe acute respiratory syndrome coronavirus 2 infection. The present thus aimed to evaluate the role of the IG count in patients hospitalized due to COVID-19 infection. The patients were predominantly infected with the alpha variant and were all unvaccinated. The IG count was measured and was found to be associated with disease severity, with patient outcomes, with the duration of hospitalization and with the development of complications. The IG count was a significantly associated with the severity of COVID-19 infection, with greater IG count values being detected in severe and critical cases. In addition, greater IG count values were associated with a longer duration of hospitalization. Furthermore, the IG count was found to be an independent prognostic biomarker of intubation and mortality in patients with COVID-19, according to multivariate logistic regression analysis, including age, the male sex and the presence of comorbidities as confounders.

BCG vaccination impacts the epigenetic landscape of progenitor cells in human bone marrow

Abstract The BCG vaccine, administered to more than 4 billion people worldwide, is designed to protect against Mycobacterium tuberculosis infection. Interestingly, clinical studies suggest that BCG also provides a degree of protection against heterologous infections, implicating other facets of the immune system apart from adaptive immunity. It has been hypothesized that BCG vaccination can leave immunological “scars” within hematopoietic stem and progenitor cells (HSPCs) that further impact downstream innate immune cell function. This is supported by studies in mice demonstrating that exposure to BCG leads to expansion and differential gene expression within hematopoietic stem cells and multipotent progenitors (HSCs/MPPs). However, very little is known about the impact of BCG vaccination on human bone marrow. Here we performed droplet-based scRNA- and scATAC-sequencing on human bone marrow aspirates from 20 healthy individuals, both before and 90 days after intradermal BCG vaccination or placebo. Over 1000 sites of differential chromatin accessibility across multiple CD34 subpopulations were present 90 days following BCG vaccination. Peaks of differential chromatin accessibility were enriched for binding of lineage determining and stress transcription factors, some of which were upregulated on the gene expression level. Expression levels of a subset of BCG-induced genes were found to significantly correlate with increased Il1b secretion of donor paired PBMCs in response to a C. albicans challenge. These data indicate that BCG vaccination re-wires transcription factor activity in human bone marrow in a way that may be linked to responses of PBMCs to secondary immune challenge with non-mycobacterial pathogens.

Non-peptidyl non-covalent cathepsin C inhibitoEEr bearing a unique thiophene-substituted pyridine: Design, structure-activity relationship and anti-inflammatory activity in vivo

Cathepsin C (Cat C) is involved in inflammation regulation by activating neutrophil serine proteases (NSPs). Therefore, Cat C is an attractive target for treatment of inflammatory diseases mediated by NSPs overactivation. In previous study, compounds 54 and 77 were reported to be the first non-peptidyl non-covalent Cat C inhibitors, with good enzyme inhibitory activity and NSPs activation inhibition, but their pharmacokinetic (PK) properties were unsatisfactory. In this study, starting from 77, after several rounds of structure-based design and modification, compound SF38, a novel Cat C inhibitor bearing a unique thiophene structure was identified, which exhibited strong inhibitory activity against Cat C (IC50 = 59.9 nM). Further mechanism study and in vivo evaluation showed that SF38 inhibited the Cat C activity in bone marrow and blood, decreased the activation of NSPs, and exhibited anti-inflammatory activity in an animal model of acute lung injury, with acceptable PK properties (F = 42.07%). These results enriched the structure-activity relationship (SAR) of Cat C inhibitor with thiophene structure characteristic, and proved the broad prospect of non-peptidyl non-covalent Cat C inhibitor.

Organ doses in preterm and full-term neonates and infants — a retrospective study on 1,064 chest radiographs

BackgroundChest radiography is the most frequent X-ray examination performed in the neonatal period. However, commonly used dosimetric entities do not describe the radiation risk sufficiently.ObjectiveThe aim of this study was to investigate selected organ doses and total body dose of chest radiographs in preterm and full-term neonates and infants.Materials and methodsIn this retrospective study, we evaluated 1,064 chest radiographs of 136 preterm and 305 full-term babies with respect to field size and centering. We calculated the entrance dose from the dose–area product. Upper and lower field borders referred to the corresponding vertebrae. We calculated individual organ doses of the thyroid, the breast, the liver and active bone marrow for each chest radiograph using the neonatal PCXMC program, a Monte Carlo program for calculating patient doses in medical X-ray examinations.ResultsThe median field size of chest radiographs ranged from 90 cm2 in preterm neonates at birth to 290 cm2 in full-term infants at the age of 6 months. Median values of entrance dose varied, depending on age, from 15 μGy to 25 μGy. The median organ doses ranged 1–20 μSv for the thyroid, 3–30 μSv for the breast, 2–20 μSv for the liver and 0.5–3.5 μSv for the bone marrow in preterm and full-term neonates and infants, respectively.ConclusionThe analysis of chest radiographs in preterm and full-term neonates and infants revealed high variability in field size. By contrast, the entrance dose varied to a minor extent. Organ dose calculations using the PCXMC program might be a valuable tool to calculate the individual radiation risk in neonates and infants.

The Programmed Death-1 Pathway Counter-Regulates Inflammation-Induced Osteoclast Activity in Clinical and Experimental Settings.

ObjectiveThe programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA.MethodsBone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores.ResultsPD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression.ConclusionThe PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.

A Pain in the Neck: An Adolescent with Neck Pain.

A Pain in the Neck: An Adolescent with Neck Pain.

Persistent Cutaneous Leishmania major Infection Promotes Infection-Adapted Myelopoiesis.

Hematopoietic stem/progenitor cells (HSPC) are responsible for the generation of most immune cells throughout the lifespan of the organism. Inflammation can activate bone marrow HSPCs, leading to enhanced myelopoiesis to replace cells, such as neutrophils, which are attracted to inflamed tissues. We have previously shown that HSPC activation promotes parasite persistence and expansion in experimental visceral leishmaniasis through the increased production of permissive monocytes. However, it is not clear if the presence of the parasite in the bone marrow was required for infection-adapted myelopoiesis. We therefore hypothesized that persistent forms of Leishmania major (cutaneous leishmaniasis) could also activate HSPCs and myeloid precursors in the C57Bl/6 mouse model of intradermal infection in the ear. The accrued influx of myeloid cells to the lesion site corresponded to an increase in myeloid-biased HSPCs in the bone marrow and spleen in mice infected with a persistent strain of L. major, together with an increase in monocytes and monocyte-derived myeloid cells in the spleen. Analysis of the bone marrow cytokine and chemokine environment revealed an attenuated type I and type II interferon response in the mice infected with the persistent strain compared to the self-healing strain, while both strains induced a rapid upregulation of myelopoietic cytokines, such as IL-1β and GM-CSF. These results demonstrate that an active infection in the bone marrow is not necessary for the induction of infection-adapted myelopoiesis, and underline the importance of considering alterations to the bone marrow output when analyzing in vivo host-pathogen interactions.

Bone Marrow-Derived RIPK3 Mediates Kidney Inflammation in Acute Kidney Injury.

Receptor-interacting protein kinase 3 (RIPK3), a component of necroptosis pathways, may have an independent role in inflammation. It has been unclear which RIPK3-expressing cells are responsible for the anti-inflammatory effect of overall Ripk3 deficiency and whether Ripk3 deficiency protects against kidney inflammation occurring in the absence of tubular cell death. We used chimeric mice with bone marrow from wild-type and Ripk3-knockout mice to explore RIPK3's contribution to kidney inflammation in the presence of folic acid-induced acute kidney injury AKI (FA-AKI) or absence of AKI and kidney cell death (as seen in systemic administration of the cytokine TNF-like weak inducer of apoptosis [TWEAK]). Tubular and interstitial cell RIPK3 expressions were increased in murine AKI. Ripk3 deficiency decreased NF-κB activation and kidney inflammation in FA-AKI but did not prevent kidney failure. In the chimeric mice, RIPK3-expressing bone marrow-derived cells were required for early inflammation in FA-AKI. The NLRP3 inflammasome was not involved in RIPK3's proinflammatory effect. Systemic TWEAK administration induced kidney inflammation in wild-type but not Ripk3-deficient mice. In cell cultures, TWEAK increased RIPK3 expression in bone marrow-derived macrophages and tubular cells. RIPK3 mediated TWEAK-induced NF-κB activation and inflammatory responses in bone marrow-derived macrophages and dendritic cells and in Jurkat T cells; however, in tubular cells, RIPK3 mediated only TWEAK-induced Il-6 expression. Furthermore, conditioned media from TWEAK-exposed wild-type macrophages, but not from Ripk3-deficient macrophages, promoted proinflammatory responses in cultured tubular cells. RIPK3 mediates kidney inflammation independently from tubular cell death. Specific targeting of bone marrow-derived RIPK3 may limit kidney inflammation without the potential adverse effects of systemic RIPK3 targeting.

CD4+ T-cell activation of bone marrow causes bone fragility and insulin resistance in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) causes an increased risk of bone fractures. However, the pathophysiology of diabetic bone fragility is not completely understood. It has been proposed that an inflammatory microenvironment in bone could be a major mechanism by inducing uncontrolled bone resorption, inadequate bone formation and consequently more porous bones. We propose that activated T-cells in the bone marrow cause a pro-inflammatory microenvironment in bone, and cause bone fragility in T2DM. We induced T2DM in C57BL/6 male mice through a hypercaloric diet rich in carbohydrates and low doses of streptozocin. In T2DM mice we inhibited systemic activation of T-cells with a fusion protein between the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 and the Fc domain of human immunoglobulin G (CTLA4-Ig). We analysed the effects of T2DM or CTLA4-Ig in lymphocyte cell subsets and antigen-presenting cells in peripheral blood and femoral bone marrow; and their effect on the metabolic phenotype, blood and bone cytokine concentration, femoral bone microarchitecture and biomechanical properties, and the number of osteoblast-like cells in the femoral endosteum. We performed a Pearson multiple correlation analysis between all variables in order to understand the global mechanism. Results demonstrated that CTLA4-Ig decreased the number of activated CD4+ T-cells in the femoral bone marrow and consequently decreased TNF-α and RANK-L concentration in bone, notably improved femoral bone microarchitecture and biomechanical properties, increased the number of osteoblast-like cells, and reduces osteoclastic activity compared to T2DM mice that did not receive the inhibitor. Interestingly, we observed that blood glucose levels and insulin resistance may be related to the increase in activated CD4+ T-cells in the bone marrow. We conclude that bone marrow activated CD4+ T-cells cause poor bone quality and insulin resistance in T2DM.

Microgels based on Infernan, a glycosaminoglycan-mimetic bacterial exopolysaccharide, as BMP-2 delivery systems

Bone Morphogenetic Protein (BMP-2) is an osteoinductive growth factor clinically used for bone regeneration. Tuneable sustained strategies for BMP-2 delivery are intensely developed to avoid severe complications related to supraphysiological doses applied. To address this issue, we investigated the ability of the bacterial exopolysaccharide (EPS) called Infernan produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus, exhibiting both glycosaminoglycan-mimetic and physical gelling properties, to efficiently bind and release the bioactive BMP-2. Two delivery systems were designed based on BMP-2 retention in either single or complex EPS-based microgels, both manufactured using a microfluidic approach. BMP-2 release kinetics were highly influenced by the ionic strength, affecting both microgel stability and growth factor/EPS binding, appearing essential for BMP-2 bioactivity. The osteogenic activity of human bone-marrow derived mesenchymal stem cells studied in vitro emphasized that Infernan microgels constitute a promising platform for BMP-2 delivery for further in vivo bone repair.

Reduced baroreflex sensitivity and increased splenic activity in patients with severe obstructive sleep apnea

Background and aimsSevere obstructive sleep apnea (OSA) is associated with an increased risk of cardiovascular disease. Experimental evidence suggests that this risk may be mediated by chronic sympathetic hyperactivation and systemic inflammation, but the precise mechanisms remain to be unraveled. Our aim was to evaluate whether severe OSA patients are characterized by increased sympathetic and hematopoietic activity, potentially driving atherosclerosis. MethodsUntreated patients with severe OSA (apnea-hypopnea index (AHI) > 30 per hour) were matched with mild OSA patients (AHI<15 & >5 per hour) according to age, sex, and body mass index. Study objectives were to assess baroreflex sensitivity (BRS) and heart-rate variability (HRV) using continuous finger blood pressure measurements, hematopoietic activity in the bone marrow and spleen, and arterial inflammation with 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT). ResultsA total of 34 subjects, 17 per group, were included in the analysis. Mean age was 60.7 ± 6.2 years, 24 (70.6%) were male. Mean AHI was 40.5 ± 12.6 per hour in the severe OSA group, and 10.5 ± 3.4 per hour in the mild OSA group. Participants with severe OSA were characterized by reduced BRS (5.7 [4.6–7.8] ms/mmHg in severe vs 8.2 [6.9–11.8] ms/mmHg in mild OSA, p = 0.033) and increased splenic activity (severe OSA 18F-FDG uptake 3.56 ± 0.77 vs mild OSA 3.01 ± 0.68; p = 0.036). HRV, bone marrow activity and arterial inflammation were comparable between groups. ConclusionsPatients with severe OSA are characterized by decreased BRS and increased splenic activity. Randomized controlled trials are warranted to assess whether OSA treatment reduces sympathetic and splenic activity.

Quantification of uptake in pelvis F-18 FLT PET-CT images using a 3D localization and segmentation CNN.

The purpose of this work was to develop and validate a deep convolutional neural network (CNN) approach for the automated pelvis segmentation in computed tomography (CT) scans to enable the quantification of active pelvic bone marrow by means of Fluorothymidine F-18 (FLT) tracer uptake measurement in positron emission tomography (PET) scans. This quantification is a critical step in calculating bone marrow dose for radiopharmaceutical therapy clinical applications as well as external beam radiationdoses. An approach for the combined localization and segmentation of the pelvis in CT volumes of varying sizes, ranging from full-body to pelvis CT scans, was developed that utilizes a novel CNN architecture in combination with a random sampling strategy. The method was validated on 34 planning CT scans and 106 full-body FLT PET-CT scans using a cross-validation strategy. Specifically, two different training and CNN application options were studied, quantitatively assessed, and statisticallycompared. The proposed method was able to successfully locate and segment the pelvis in all test cases. On all data sets, an average Dice coefficient of 0.9396 0.0182 or better was achieved. The relative tracer uptake measurement error ranged between 0.065% and 0.204%. The proposed approach is time-efficient and shows a reduction in runtime of up to 95% compared to a standard U-Net-based approach without a localizationcomponent. The proposed method enables the efficient calculation of FLT uptake in the pelvis. Thus, it represents a valuable tool to facilitate bone marrow preserving adaptive radiation therapy and radiopharmaceutical dose calculation. Furthermore, the method can be adapted to process other bone structures as well asorgans.

Emilin-2 is a component of bone marrow extracellular matrix regulating mesenchymal stem cell differentiation and hematopoietic progenitors

BackgroundDissection of mechanisms involved in the regulation of bone marrow microenvironment through cell–cell and cell–matrix contacts is essential for the detailed understanding of processes underlying bone marrow activities both under physiological conditions and in hematologic malignancies. Here we describe Emilin-2 as an abundant extracellular matrix component of bone marrow stroma.MethodsImmunodetection of Emilin-2 was performed in bone marrow sections of mice from 30 days to 6 months of age. Emilin-2 expression was monitored in vitro in primary and mesenchymal stem cell lines under undifferentiated and adipogenic conditions. Hematopoietic stem cells and progenitors in bone marrow of 3- to 10-month-old wild-type and Emilin-2 null mice were analyzed by flow cytometry.ResultsEmilin-2 is deposited in bone marrow extracellular matrix in an age-dependent manner, forming a meshwork that extends from compact bone boundaries to the central trabecular regions. Emilin-2 is expressed and secreted by both primary and immortalized bone marrow mesenchymal stem cells, exerting an inhibitory action in adipogenic differentiation. In vivo Emilin-2 deficiency impairs the frequency of hematopoietic stem/progenitor cells in bone marrow during aging.ConclusionOur data provide new insights in the contribution of bone marrow extracellular matrix microenvironment in the regulation of stem cell niches and hematopoietic progenitor differentiation.

Study of the effect of hypothyroidism and thyrotoxicosis on the proliferative activity of the red bone marrow in experimental animals with an implanted Akatol tumor

Study of the effect of hypothyroidism and thyrotoxicosis on the proliferative activity of the red bone marrow in experimental animals with an implanted Akatol tumor

Preparation and Inflammasome Activation of Murine Bone Marrow-Derived and Resident Peritoneal Macrophages.

Cholera toxin (CT), secreted by Vibrio cholerae, not only causes cholera but also functions as an immune adjuvant. Macrophages, which respond to a variety of immune adjuvants, are quite heterogenous and their development and function depend on the tissues where they are localized. We have characterized the effects of the B subunit of CT (CTB) on two types of murine macrophages, that is, bone marrow-derived macrophages (BMMs) and resident peritoneal macrophages (rPMs). CTB could induce production of interleukin-1β (IL-1β) from both macrophages in synergy with lipopolysaccharides. However, underlying molecular mechanisms for IL-1β induction were different. Here, we describe the protocols for preparation and stimulation of BMMs and rPMs.

Use of &lt;i&gt;Escherichia coli&lt;/i&gt; expression system for analyzing kinase motifs

Sixty-eight human tyrosine kinases were cloned into the Escherichia coli expression vector pET21a(+) and 48 of them were successfully expressed in the BL21(DE3) strain. Among them, 18 kinases were activated in the bacterial cells by autophosphorylation and they phosphorylated endogenous proteins. Since E. coli expression systems do not usually enable post-translational modifications, the in vivo phosphorylation should be due to the tyrosine kinase expressed. In this study, we attempted to use the bacterial expression system as a site for the kinase reaction to determine the substrate sequence preference of a certain kinase, namely, a phosphorylation motif. As a first example, phosphoproteomic analysis of E. coli expressing active bone marrow tyrosine kinase gene in chromosome X protein (BMX) was performed to extract the phosphorylation motifs. The motifs were compared with that obtained from Kinapple, the database of in vitro human kinome profiling, and common features rich in acidic amino acids were found. Furthermore, the phosphorylation state of expressed BMX was analyzed. Phos-tag SDS-PAGE in conjunction with alanine scans of the phosphorylation sites found by mass spectrometry revealed that Y224, Y234, and Y566 are predominantly phosphorylated. Moreover, phosphorylation at S453 and T572 was implicated in activation in E. coli, whereas these sites did not affect activity in 293 cells. These results suggest that, although the bacterial system does not fully reflect the physiological properties of the kinase, it is a simple experimental method that can infer the substrate preference of tyrosine kinases similar to that observed with substrates of human origin.

Assessing Bone Marrow Activity with [18F]FLT PET in Patients with Essential Thrombocythemia and Prefibrotic Myelofibrosis: A Proof of Concept.

Objectives: This study aims to assess the value of FLT-PET as a non-invasive tool to differentiate between patients with ET and Pre-PMF. This study is a pilot study to have a proof of concept only. Methods: This is a prospective, interventional study where a total of 12 patients were included. Each patient underwent FLT PET imaging as well as bone marrow examination (gold standard). In addition, semi-quantitative (SUVmax and SUVmean) measurements of FLT uptake in the liver, spleen, and Lspine, SUVmean, as well as the Total Lesion Glycolysis (TLG) of the Lspine were performed. Results from the two patient cohorts were compared using = Kruskal-Wallis statistical test. A P-value of <.05 is considered to be statistically significant. Results: The differences in FLT SUVmax and SUVmean measurements in the three organs (liver, spleen, and LSpine) between the ET and Pre-PMF patients were not statistically significant (P > .05). In contrast, TLG measurements in the LSpine were statistically different (P = .013), and therefore, compared to gold standard bone marrow results, TLG can separate ET and Pre-PMF patients. Conclusion: This study is a proof of concept about the potential to discriminate between ET and pre-PMF patients in a non-invasive way. TLG of the LSpine in FLT PET images is a potential quantitative parameter to distinguish between ET and pre-PMF patients.

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow

Low-LET photon radiation-induced persistent alterations in bone marrow (BM) cells are well documented in total-body irradiated (TBI) rodents and also among radiotherapy patients. However, the late effects of protons and high-LET heavy-ion radiation on BM cells and its implications in osteoclastogenesis are not fully understood. Therefore, C57BL6/J female mice (8 weeks; n = 10/group) were irradiated to sham, and 1 Gy of the proton (0.22 keV/μm), or high-LET 56Fe-ions (148 keV/μm) and at 60 d post-exposure, mice were sacrificed and femur sections were obtained for histological, cellular and molecular analysis. Cell proliferation (PCNA), cell death (active caspase-3), senescence (p16), osteoclast (RANK), osteoblast (OPG), osteoblast progenitor (c-Kit), and osteoclastogenesis-associated secretory factors (like RANKL) were assessed using immunostaining. While no change in cell proliferation and apoptosis between control and irradiated groups was noted, the number of BM megakaryocytes was significantly reduced in irradiated mice at 60 d post-exposure. A remarkable increase in p16 positive cells indicated a persistent increase in cell senescence, whereas increased RANKL/OPG ratio, reductions in the number of osteoblast progenitor cells, and osteocalcin provided clear evidence that exposure to both proton and 56Fe-ions promotes pro-osteoclastogenic activity in BM. Among irradiated groups, 56Fe-induced alterations in the BM cellularity and osteoclastogenesis were significantly greater than the protons that demonstrated a radiation quality-dependent effect. This study has implications in understanding the role of IR-induced late changes in the BM cells and its involvement in bone degeneration among deep-space astronauts, and also in patients undergoing proton or heavy-ion radiotherapy.

The cathelicidin CATH-2 efficiently neutralizes LPS- and E. coli-induced activation of porcine bone marrow derived macrophages

Infectious diseases in pigs cause monetary loss to farmers and pose a zoonotic risk. Therefore, it is important to obtain more porcine specific immunological knowledge as a measure to protect against infectious diseases, for example by exploring immunomodulators that are usable as vaccine adjuvants. Cathelicidins are a class of host defence peptides (HDPs) able to directly kill microbes as well as exert a diverse range of effects on the immune system. The peptides have shown promise as immunomodulatory peptides in many applications, including vaccines. However, it is currently unknown what the precise effect of these peptides is on porcine immune cells and whether peptides of other species might also have a strong immunomodulatory effect on porcine macrophages. Mononuclear bone marrow cells of pigs, aged 5–6 months, were cultured into M1 or M2 macrophages and stimulated with LPS or whole bacteria in the presence of host defence peptides (HDPs).CATH-2 and LL-37 strongly inhibited LPS-induced activation of M1 macrophages, the inhibition of LPS-induced activation of M2 macrophages by HDPs was milder, showing that the peptides have selective effects on different cell types. Upon stimulation with whole bacteria, only CATH-2 could effectively inhibit macrophage activation, showing the potent anti-inflammatory potential of this peptide. These results show that porcine peptides are not necessarily the most active in a porcine system, and that CATH-2 is effective in a porcine system as an anti-inflammatory immune modulator, which can be used, for example, in inactivated pathogen vaccines.

Bone Marrow-Resident Vδ1 T Cells Co-express TIGIT With PD-1, TIM-3 or CD39 in AML and Myeloma

Background: γδ T cells represent a unique T cell subpopulation due to their ability to recognize cancer cells in a T cell receptor- (TCR) dependent manner, but also in a non-major histocompatibility complex- (MHC) restricted way via natural killer receptors (NKRs). Endowed with these features, they represent attractive effectors for immuno-therapeutic strategies with a better safety profile and a more favorable anti-tumor efficacy in comparison to conventional αβ T cells. Also, remarkable progress has been achieved re-activating exhausted T lymphocytes with inhibitors of co-regulatory receptors e.g., programmed cell death protein 1 (PD-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT) and of the adenosine pathway (CD39, CD73). Regarding γδ T cells, little evidence is available. This study aimed to immunophenotypically characterize γδ T cells from patients with diagnosed acute myeloid leukemia (AML) in comparison to patients with multiple myeloma (MM) and healthy donors (HD).Methods: The frequency, differentiation, activation, and exhaustion status of bone marrow- (BM) derived γδ T cells from patients with AML (n = 10) and MM (n = 11) were assessed in comparison to corresponding CD4+ and CD8+ T cells and peripheral blood- (PB) derived γδ T cells from HDs (n = 16) using multiparameter flow cytometry.Results: BM-infiltrating Vδ1 T cells showed an increased terminally differentiated cell population (TEMRAs) in AML and MM in comparison to HDs with an aberrant subpopulation of CD27−CD45RA++ cells. TIGIT, PD-1, TIM-3, and CD39 were more frequently expressed by γδ T cells in comparison to the corresponding CD4+ T cell population, with expression levels that were similar to that on CD8+ effector cells in both hematologic malignancies. In comparison to Vδ2 T cells, the increased frequency of PD-1+-, TIGIT+-, TIM-3+, and CD39+ cells was specifically observed on Vδ1 T cells and related to the TEMRA Vδ1 population with a significant co-expression of PD-1 and TIM-3 together with TIGIT.Conclusion: Our results revealed that BM-resident γδ T cells in AML and MM express TIGIT, PD-1, TIM-3 and CD39. As effector population for autologous and allogeneic strategies, inhibition of co-inhibitory receptors on especially Vδ1 γδ T cells may lead to re-invigoration that could further increase their cytotoxic potential.