Stimulation Of Leukocytes Research Articles

Quantiferon Monitor Testing Sheds Light on Immune System Disparities between Multiple Sclerosis Patients and Healthy Individuals.

The aim of this study was to conduct QuantiFERON Monitor (QFM) testing in patients with multiple sclerosis (MS), which is used to monitor the state of the immune system through the non-specific stimulation of leukocytes followed by determining the level of interferon-gamma (IFN-γ) released from activated cells. Additionally, we tested the level of selected cytokines (IFN-α, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-15, IL-33, VEGF) from stimulated blood samples to further understand the immune response. This study builds upon a previously published study, utilizing activated serum samples that were initially used for IFN-γ determination. However, our current focus shifts from IFN-γ to exploring other cytokines that could provide further insights into the immune response. A screening was conducted using Luminex technology, which yielded promising results. These results were then further elaborated upon using ELISA to provide a more detailed understanding of the cytokine profiles involved. This study, conducted from August 2019 to June 2023, included 280 participants: 98 RRMS patients treated with fingolimod (fMS), 96 untreated patients with progressive MS (pMS), and 86 healthy controls (HC). Our results include Violin plots showing elevated IL-1α in pMS and fMS. Statistical analysis indicated significant differences in the interleukin levels between groups, with IL-1ra and age as key predictors in differentiating HC from pMS and IL-1ra, IL-1α, age, and EDSS in distinguishing pMS from fMS. These findings suggest cytokines' potential as biomarkers in MS progression and treatment response.

Green and red macroalgae extracts show antibacterial effects and induce innate immune responses in Nile tilapia and rainbow trout in vitro

Green macroalgae (Ulva sp.) and red macroalgae (Solieria sp.) were processed to produce eight different crude extracts enriched for marine sulphated polysaccharides (MSP). While all extracts showed direct anti-bacterial effects to some degree, two red algal extracts, in particular, showed high activity against several pathogenic fish bacteria in vitro. Stimulation of head kidney leukocytes (HKLs) in vitro with MSP-rich extracts showed fish species specific differences. In Nile tilapia, HKLs showed a dose dependent production of reactive oxygen species (ROS) following stimulation with Ulva-derived extracts, while stimulation with Solieria-derived extracts did not induce ROS production. In rainbow trout, HKLs showed relatively high reactive ROS potential and Solieria-derived extracts could induce significant ROS production, albeit without a clear dose dependent response. Gene expression of in vitro stimulated HKLs showed a clear induction of most cytokines measured (il1b, il10, tnfa, ifny, il12p40), with differences between species. Although cytokine gene expressions were more prominently induced in HKLs of Nile tilapia than in HKLs of rainbow trout, immunomodulatory effects of both, Ulva- and Solieria-derived extracts, always appeared evident. It is likely that HKLs have the ability to sense presence of MSP, via so far uncharacterized pattern recognition receptors which have known downstream signalling pathways. Expression of some genes associated with Toll-like Receptor (TLR) signalling, such as irak1 and especially the expression of genes associated with C-type Lectin Receptor (CLR) signalling, such as card9 and bcl10 were evident following stimulation with MSP-rich extracts. Possibly, immunomodulatory effects of MSP could be regulated by at least a CLR-mediated signalling, at least in Nile tilapia, for which most if not all red algae and green algae extracts induced changes in gene expression of card9 and bcl10. Overall, induction of ROS production and gene expression read-outs suggest immunomodulatory effects of MSP-rich extracts derived from green algae (Ulva sp.) and red algae (Solieria sp.), at least in vitro. The observed effects suggest clear fish species specific differences between the effects of MSP-rich extracts.

What is the role of the neutrophil extracellular traps in the cardiovascular disease burden associated with hemodialysis bioincompatibility?

Despite significant progress in dialysis modalities, intermittent renal replacement therapy remains an "unphysiological" treatment that imperfectly corrects uremic disorders and may lead to low-grade chronic inflammation, neutrophil activation, and oxidative stress due to repetitive blood/membrane interactions contributing to the "remaining uremic syndrome" and cardiovascular disease burden of hemodialysis patients. Understanding dialysis bioincompatibility pathways still remains a clinical and biochemical challenge. Indeed, surrogate biomarkers of inflammation including C-reactive protein could not discriminate between all components involved in these complex pathways. A few examples may serve to illustrate the case. Cytokine release during dialysis sessions may be underestimated due to their removal using high-flux dialysis or hemodiafiltration modalities. Complement activation is recognized as a key event of bioincompatibility. However, it appears as an early and transient event with anaphylatoxin level normalization at the end of the dialysis session. Complement activation is generally assumed to trigger leukocyte stimulation leading to proinflammatory mediators' secretion and oxidative burst. In addition to being part of the innate immune response involved in eliminating physically and enzymatically microbes, the formation of Neutrophil Extracellular Traps (NETs), known as NETosis, has been recently identified as a major harmful component in a wide range of pathologies associated with inflammatory processes. NETs result from the neutrophil degranulation induced by reactive oxygen species overproduction via NADPH oxidase and consist of modified chromatin decorated with serine proteases, elastase, bactericidal proteins, and myeloperoxidase (MPO) that produces hypochlorite anion. Currently, NETosis remains poorly investigated as a sensitive and integrated marker of bioincompatibility in dialysis. Only scarce data could be found in the literature. Oxidative burst and NADPH oxidase activation are well-known events in the bioincompatibility phenomenon. NET byproducts such as elastase, MPO, and circulating DNA have been reported to be increased in dialysis patients more specifically during dialysis sessions, and were identified as predictors of poor outcomes. As NETs and MPO could be taken up by endothelium, NETs could be considered as a vascular memory of intermittent bioincompatibility phenomenon. In this working hypothesis article, we summarized the puzzle pieces showing the involvement of NET formation during hemodialysis and postulated that NETosis may act as a disease modifier and may contribute to the comorbid burden associated with dialysis bioincompatibility.

Developing a Prolamin-Based Gel for Food Packaging: In-Vitro Assessment of Cytocompatibility.

Growing environmental concerns drive efforts to reduce packaging waste by adopting biodegradable polymers, coatings, and films. However, biodegradable materials used in packaging face challenges related to barrier properties, mechanical strength, and processing compatibility. A composite gel was developed using biodegradable compounds (prolamin, d-mannose, citric acid), as a coating to increase the oxygen barrier of food packaging materials. To improve gel stability and mechanical properties, the gels were physically cross-linked with particles synthesized from tetraethyl orthosilicate and tetramethyl orthosilicate precursors. Additionally, biocompatibility assessments were performed on human keratinocytes and fibroblasts, demonstrating the safety of the gels for consumer contact. The gel properties were characterized, including molecular structure, morphology, and topography. Biocompatibility of the gels was assessed using bioluminescent ATP assay to detect cell viability, lactate dehydrogenase assay to determine cell cytotoxicity, and a leukocyte stimulation test to detect inflammatory potential. A composite gel with strong oxygen barrier properties in low-humidity environments was prepared. Increasing the silane precursor to 50 wt% during gel preparation slowed degradation in water. The addition of citric acid decreased gel solubility. However, higher precursor amounts increased surface roughness, making the gel more brittle yet mechanically resistant. The increase of precursor in the gel also increased gel viscosity. Importantly, the gels showed no cytotoxicity on human keratinocytes or fibroblasts and had no inflammatory effects on leukocytes. This composite gel holds promise for oxygen barrier food packaging and is safe for consumer contact. Further research should focus on optimizing the stability of the oxygen barrier in humid environments and investigate the potential sensitizing effects of biodegradable materials on consumers.

Human Whole Blood Interactions with Craniomaxillofacial Reconstruction Materials: Exploring In Vitro the Role of Blood Cascades and Leukocytes in Early Healing Events.

The present study investigated early interactions between three alloplastic materials (calcium phosphate (CaP), titanium alloy (Ti), and polyetheretherketone (PEEK) with human whole blood using an established in vitro slide chamber model. After 60 min of contact with blood, coagulation (thrombin-antithrombin complexes, TAT) was initiated on all test materials (Ti > PEEK > CaP), with a significant increase only for Ti. All materials showed increased contact activation, with the KK-AT complex significantly increasing for CaP (p < 0.001), Ti (p < 0.01), and PEEK (p < 0.01) while only CaP demonstrated a notable rise in KK-C1INH production (p < 0.01). The complement system had significant activation across all materials, with CaP (p < 0.0001, p < 0.0001) generating the most pronounced levels of C3a and sC5b-9, followed by Ti (p < 0.001, p < 0.001) and lastly, PEEK (p < 0.001, p < 0.01). This activation correlated with leukocyte stimulation, particularly myeloperoxidase release. Consequently, the complement system may assume a more significant role in the early stages post implantation in response to CaP materials than previously recognized. Activation of the complement system and the inevitable activation of leukocytes might provide a more favorable environment for tissue remodeling and repair than has been traditionally acknowledged. While these findings are limited to the early blood response, complement and leukocyte activation suggest improved healing outcomes, which may impact long-term clinical outcomes.

Pilot Study of the Ex Vivo Blood Leukocytes’ Proteomic Response to Prednisone Stimulation in Corticosteroid-responsive Asthma

Background: Leukocytes are key cellular effectors of inflammation in asthma and understanding their function in this disease is of crucial importance. Blood leukocytes reflect the actions of their counterparts in the lungs and they can be obtained through minimal invasive procedures as part of the peripheral blood. Objective: The aim of the study was to identify proteins in blood leukocyte proteomes that respond to ex vivo treatment by prednisone in order to pinpoint candidates for predictive biomarkers in corticosteroid- responsive asthma. Methods: The study included five children diagnosed with asthma and five healthy children. After the ex vivo treatment of blood samples with prednisone, lysis of erythrocytes was performed and proteins were extracted from the remaining leukocytes by ultrasonic disintegration. Protein extracts were analyzed by reversed phase nano-liquidchromatography–tandem mass spectrometry (LC–MS/MS). Results: The stimulation of asthmatics' leukocytes with prednisone has led to an increase in the levels of FYB (fold change 3.4) and LYZ (fold change 2.2) with a statistical significance of p&lt;0.005. The two proteins with expressions significantly altered upon the prednisone treatment should be further explored as tools to evaluate the patient's response before therapy administration, especially when lung function measurements are not possible, as is the case with young pediatric patients. Conclusion: The approach that entails ex vivo response of blood leukocytes to therapeutics can facilitate asthma management and help overcome the need for therapeutic adjustments in a clinical setting.

Immune response of European sea bass (Dicentrarchus labrax L.) against combination of antigens from three different pathogens

Three of the most important diseases of Mediterranean intensive European sea bass farming are, viral nervous necrosis (VNN) caused by the red grouper nervous necrosis virus (RGNNV) genotype of b-nodavirus, photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp) and vibriosis caused mainly by the O1 serotype of Vibrio anguillarum (VaO1). Prevention against these diseases is performed through vaccination with a monovalent vaccine against the viral disease and, usually, with bivalent vaccines against the bacterial diseases. However, it is very difficult to program two vaccinations during the same season for the same fish stock and producers are forced to either vaccinate for the viral or the bacterial diseases or to perform double vaccination with both vaccines, without any prior knowledge on any interactions that may occur due to the plethora of antigens (Ag) injected. Ideally, therefore, a trivalent vaccine should be developed against all three diseases. The objective of this work was to analyse the immune response of sea bass against combinations of Ags from all three pathogens, namely viral particles, Phdp whole cells (WC), lipopolysaccharide (LPS), capsular polysaccharide (CPS) and extracellular products (ECPs) and VaO1 WC and ECPs in respect to the identification of any phenomena of immunodominance/immunosuppression between Ags with a view to select candidate Ags for inclusion in a trivalent vaccine formulation. Eight triplicate groups of fish were immunized with different combinations of the aforementioned Ags and another triplicate group served as negative control. Blood serum was isolated at various time-points post-immunization for the measurement of specific antibodies against each Ag and, in addition, leucocytes were isolated at day 29 post-immunization for analysis of various cellular activities. Results indicated that best levels of specific a-NNV virus antibodies (Abs) were produced when VaO1 ECPs were not included in the Ag combinations, in contrast to the leucocytes proliferation assay where best stimulation against NNV Ags was measured when VaO1 ECPs were present in Ag combinations. VaO1 ECPs apparently is a strong immunogen for both humoral and cellular responses but suppresses immunological reactions against the other Ags.VaO1 WC, Phdp LPS and ECPs raised good humoral immune responses in the groups with best responses against VNN Ags, but only VaO1 WC and Phdp ECPs provided good stimulation of leucocytes, with Phdp WC and CPS effecting either similar stimulation with untrained leucocytes (control groups) or down-stimulation. Results are discussed with a view to select Ags from all three pathogens for inclusion in trivalent vaccine against all three pathogens.

Cylindropuntia cholla aqueous root rich in phytosterols enhanced immune response and antimicrobial activity in tilapia Oreochromis niloticus leukocytes

“Cacti” are rich sources of phytochemicals with antioxidant activity, and their use is mainly focused on infusions in traditional medicine in Mexico. This study characterizes the chemical compounds found in Cylindropuntia cholla root by gas chromatography coupled to mass spectrometry (GC-MS) and determines the total content of polyphenols and flavonoids, as well as their antioxidant capacity. The immunostimulatory effect of aqueous C. cholla root extract (ACcr) was evaluated at concentrations of 50, 250, 500, and 1000 μg/mL in Tilapia peripheral blood leukocytes. The results obtained by the GC-MS analysis revealed the presence of phenolic acids, flavonoid and phytosterol derivatives as β-sitosterol and campesterol. The determination of the total polyphenol and flavonoid contents indicated that ACcr is abundant in polyphenols, showing an anti-radical capacity of scavenging free radicals, such as those of hydroxyl and superoxide, as well as an increase in lipid peroxidation inhibition capacity. Stimulation of tilapia leukocytes resulted in the increase of its phagocytic activity, respiratory burst, nitric oxide production, and superoxide dismutase activity. Finally, the results obtained for the first time allowed establishing the chemical profile of ACcr and its antimicrobial activity against three important pathogenic bacteria. The potential of this root is indicated as an additive in formulating antioxidant and immunostimulant supplements for the aquaculture and pharmaceutical industry.

A physical wiring diagram for the human immune system

The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention.

Regulation of Neutrophil NADPH Oxidase, NOX2: A Crucial Effector in Neutrophil Phenotype and Function.

Reactive oxygen species (ROS), produced by the phagocyte NADPH oxidase, NOX2, are involved in many leukocyte functions. An excessive or inappropriate ROS production can lead to oxidative stress and tissue damage. On the other hand, an absence of ROS production due to a lack of a functional NADPH oxidase is associated with recurrent infections as well as inflammation disorders. Thus, it is clear that the enzyme NADPH oxidase must be tightly regulated. The NOX2 complex bears both membrane and cytosolic subunits. The membrane subunits constitute the flavocytochrome b 558, consisting of gp91phox (Nox2) and p22phox subunits. The cytosolic subunits form a complex in resting cells and are made of three subunits (p47phox, p40phox, p67phox). Upon leukocyte stimulation, the cytosolic subunits and the small GTPase Rac assemble with the flavocytochrome b 558 in order to make a functional complex. Depending on the stimulus, the NADPH oxidase can assemble either at the phagosomal membrane or at the plasma membrane. Many studies have explored NOX2 activation; however, how this activation is sustained and regulated is still not completely clear. Here we review the multiple roles of NOX2 in neutrophil functions, with a focus on description of its components and their assembly mechanisms. We then explain the role of energy metabolism and phosphoinositides in regulating NADPH oxidase activity. In particular, we discuss: 1) the link between metabolic pathways and NOX2 activity regulation through neutrophil activation and the level of released ROS, and 2) the role of membrane phosphoinositides in controlling the duration of NOX2 activity.

Protective Effect against Neosporosis Induced by Intranasal Immunization with Neospora caninum Membrane Antigens Plus Carbomer-Based Adjuvant.

Neospora caninum is an obligate intracellular protozoan responsible for abortion and stillbirths in cattle. We previously developed a mucosal vaccination approach using N. caninum membrane proteins and CpG adjuvant that conferred long-term protection against neosporosis in mice. Here, we have extended this approach by alternatively using the carbomer-based adjuvant Carbigen™ in the immunizing preparation. Immunized mice presented higher proportions and numbers of memory CD4+ and CD8+ T cells. Stimulation of spleen, lungs and liver leukocytes with parasite antigens induced a marked production of IFN-γ and IL-17A and, less markedly, IL-4. This balanced response was also evident in that both parasite-specific IgG1 and IgG2c were raised by immunization, together with specific intestinal IgA. Upon intraperitoneal infection with N. caninum, immunized mice presented lower parasitic burdens than sham-immunized controls. In the infected immunized mice, memory CD4+ T cells predominantly expressed T-bet and RORγt, and CD8+ T cells expressing T-bet were found increased. While spleen, lungs and liver leukocytes of both immunized and sham-immunized infected animals produced high amounts of IFN-γ, only the cells from immunized mice responded with high IL-17A production. Since in cattle both IFN-γ and IL-17A have been associated with protective mechanisms against N. caninum infection, the elicited cytokine profile obtained using CarbigenTM as adjuvant indicates that it could be worth exploring for bovine neosporosis vaccination.

Septilin, A Polyherbal Formulation Against the Immunosuppression Induced by Cyclophosphamide and Cisplatin in Swiss Albino Mice

Background: Septilin (Spt) is an ayurvedic drug formulation from Himalaya Drug Company and is well-known for its antibacterial, anti-inflammatory and immunomodulatory activities. Interest in the use of medicinal plants and herbal medicine as immunomodulators has currently been the subject of scientific investigation worldwide. Cyclophosphamide (CP) and Cisplatin (Csp) are widely used chemotherapeutic drugs and are known for their immunosuppressive effects. Methods: The present study evaluated the immune-stimulating activity of Spt (125, 250 and 500 mg/kg; PO/7 days) against CP (50 mg/kg) and Csp (10 mg/kg) induced immunosuppression in mice sensitized with sheep Red Blood Cells (RBC) by measuring Hemagglutination Antibody (HA) titre values and by determining the haematological parameters, such as Haemoglobin (Hb) content, White Blood Cells (WBC), RBC and platelet counts. Thymus index and differential counts of leukocytes were also determined. Results: Upon HA assay, the titre was significantly decreased in CP (59.40%) and Csp (62.16%) in the treatment groups (P&lt;0.001). An increase in the HA titre value in Spt-treated mice showed the stimulation of humoral immune response (P&lt;0.001). The results of haematological study in Spt-treated mice indicated stimulation of total leukocytes, RBC and platelet counts. Moreover, Spt treatment prior to the administration of CP and Csp prevented the loss of body weight and minimized their adverse effect on the mice thymus. Conclusion: Our experimental evidence suggests the immunostimulatory potency of Spt against the immunosuppression induced by chemotherapeutic drugs in mice. The study results are comparable with the immune-potentiating effects of standard immunomodulatory drug Levamisole (Lev). Hence, Spt may be used as an adjuvant to obviate the immune suppression induced by chemotherapeutic medications.

A Possible Mechanism of Action of 17α-Hydroxyprogesterone Caproate: Enhanced IL-10 Production.

The rate of recurrent spontaneous preterm birth (PTB) was reduced by 33% in the Maternal-Fetal Medicine Unit (MFMU) Network trial of 17α-hydroxyprogesterone caproate (17-OHPC), but the mechanism of action, 17 years later, remains elusive. The robustness of the interleukin-10 (IL-10) response to lipopolysaccharide (LPS) stimulation of leukocytes in pregnant women with a prior PTB correlates with gestational age at delivery. This study sought to determine if there is a relationship between the concentration of 17-OHPC and response to LPS stimulation. We performed a secondary analysis of data from the Omega-3 MFMU trial which evaluated the effectiveness of omega-3 fatty acid supplementation in reducing recurrent PTB. We utilized previously characterized data from a subanalyses of the Omega-3 trial of IL-10 and tumor necrosis factor alpha (TNF-α) levels from peripheral blood mononuclear cells stimulated with LPS. Blood was obtained from enrolled women at 16 to 22 weeks' gestation (baseline) and 25 to 28 weeks' gestation (posttreatment). All women received 17-OHPC and plasma 17-OHPC concentrations were measured at 25 to 28 weeks' gestation. We analyzed these data to determine if there was a relationship between 17-OHPC concentration and cytokine production. We then performed an in vitro study to determine if 17-OHPC could directly alter cytokine production by THP-1-derived macrophages. In the clinical samples, we found that 17-OHPC plasma concentrations were correlated with the quantity of the LPS-stimulated production of IL-10. TNF-α production after LPS stimulation was unrelated to 17-OHPC concentration. In the in vitro study, we demonstrate a 17-OHPC concentration dependent increase in IL-10 production. In women receiving 17-OHPC for PTB prevention, we demonstrate a relationship between plasma 17-OHPC and LPS-stimulated IL-10 production by circulating leukocytes. We also demonstrate that, in vitro, 17-OHPC treatment affects IL-10 production by LPS-stimulated macrophages. Collectively, these findings support an immunomodulatory mechanism of action of 17-OHPC in the prevention of recurrent PTB. · 17-OHPC plasma concentrations and LPS-stimulated IL-10 levels correlate in clinical samples in women at risk for recurrent preterm birth.. · 17-OHPC can modulate the response of LPS-stimulated macrophages to increase IL-10 production.. · There was no relationship between TNF-α and plasma concentration of 17-OHPC in clinical samples or in vitro..

Biochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy.

Biochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte’s specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.

Cell-mediated immune response against mycolic acids of Mycobacteroides salmoniphilum in rainbow trout Oncorhynchus mykiss

Mycobacteriosis caused by Mycobacterium spp. causes economic damages to the world aquaculture industry. In mammals, mycolic acids contained in the cell wall of Mycobacterium spp. are presented by CD1b molecule as lipid antigens and induce cell-mediated immunity (CMI). Here, we investigated CMI responses against the mycolic acids of Mycobacterioides salmoniphilum in a CD1-lacking teleost fish, rainbow trout. After stimulation of trout leukocytes with mycolic acids, the number and percentage of CD8α+ T cells increased. Fish immunized with mycolic acids showed an up-regulation of IFN-γ. Further, in vitro re-stimulation of leukocytes derived from immunized fish resulted in proliferation of CD8α+ cells. These data suggest that mycolic acids are recognized as lipid antigens resulting in an activation of rainbow trout CD8α+ cells and up-regulation of the Th1 cytokine IFN-γ. The mycolic acids are promising candidates for vaccines to activate CD8α+ T cells against fish mycobacteriosis.

Poor Prognosis of CD5 Positive with Non Germinal Center Type of Diffuse Large B Cell Lymphoma Especially Having the Aberration of 19q13

Introduction ; One of biological molecule markers, CD5 positive (CD5+) DLBCL has been recognized as one of the poor prognostic factors. Almost all CD5+DLBCL cases have higher age, aggressive clinical features, poor performance status (PS), and high LDH level compared to CD5 negative (CD5-) DLBCL. These clinical features are close to ABC type DLBCL and also some gene expression profiling detected the similar point of CD5+ DLBCL with ABC type of DLBCL, such as down-regulation of ECUM genes. Various chromosomal aberrations and polyploidy also have been detected in CD5+ DLBCL, although, specific features of karyotype or markers of gene expression have not been identified. In previous data, we presented the poor prognosis of CD5+non germinal center (NGC) type of DLBCL compared to CD5+ germinal center (GC) type DLBCL and CD5-DLBCL, furthermore additional 19q13 were frequently exhibited in CD5+NGC DLBCL (ASH 2015, poster). In this time we added the more analysis of G-band analysis of DLBCL and CGH array of CD5+NGC with additional 19q13 and analyzed the prognostic factor of CD5+NGC DLBCL.Methods and Results ; We analyzed our treated 373 DLBCL patients by R-CHOP during 2005-2015 in our institute retrospectively. This protocol was approved by our Institutional Review Board and Genomic review Board. All cases were classified depend on CD5+ or CD5- expression by immuno-staining and/or flowcytometry from tumor-biopsy specimen and furthermore sub-divided to GCB or NGC by immuno-staining, as CD10, bcl-6, and MUM-1. 350 cases were encluded, then CD5+ were 41 (41/350; 11.7%) CD5+NGC type were 30 (30/41; 73.2%). 3.5 year-PFS was as follows, CD5-GCB; 84.0% (95%CI 77.4-88.8%), CD5+GCB; 65.9% (95%CI 56.2-74.0%), CD5-NGC; 81.8% (95%CI 44.7-95.1%), and CD5+NGC; 40.6% (95%CI 22.5-58.0%), respectively. The 3.5 year-OS was as follows, CD5-GCB; 85.0% (95%CI 78.4-89.8%), CD5+GCB; 79.7% (95%CI 70.9-86.1%), CD5-NGC; 90.9% (95%CI 50.8-98.7%), and CD5+NGC; 44.5% (95%CI 25.2-62.1%), respectively. CD5+NGC type of DLBCL were demonstrated the worst prognosis in both PFS (P=0.00104) and OS (P <0.001), respectively. By the multivariate analysis of prognostic factors, CD5+ (HR; 2.180, 95%CI; 1.263-3.764, P=0.0051) and IPI (HR; 1.678, 95%CI; 1.151-2.449, P=0.0071) were estimated as individual factors for OS. CD5+NGC type of DLCBL frequently showed the chromosomal aberration of additional 19q13 (7/13 cases; 53.8%). additional 19q13 was also showed only one case of CD5-NGC type of DLBCL. Six cases (6/7; 85.7%) in CD5+NGC had disease progression (two cases relapsed after CR and four had progression during R-CHOP therapy) and followed by death. CGH analysis was performed with the five cases having additional 19q13 in CD5+NGC type. All cases exhibited the aberration of 19q13.41-42 lesions. Three cases showed wide gene amplification in 19q13.32-q13.42. Three cases showed gene loss at the 19q13.41 or 19q13.42. Regarding other chromosomes, all five cases showed gene loss at 1p31.3 and gain at 1q25.1, 2p22.1, 2p22.3, 4p16.1, 4p15.1, 7q33, 12p13.2, 14q32.33.Discussion and Conclusion ;Our study showed significant poor prognosis CD5+NGC type of DLBCL. The gene aberration of 19q13 was frequently expressed in CD5+NGC type of DLBCL and it suggested the relationships between aberration of 19q13 and the poor prognosis. Several study also demonstrated the relationships between aberration of 19q13 and the poor prognosis of the DLBCL. Our CGH analysis demonstrated the aberration of 19q13.41-42 lesions. Already known representative CNVs in this area include CYP2A, SIGLEC14, and LILRA. Those candidate genes have reported to have the relationships with stimulation of leukocyte or lymphoid cell signaling. The further investigation of gene aberration regarding to additional 19q13 might be one of the procedure to overcome the tolerability for the treatment of CD5+NGC DLBCL. From our data we could consider that it is important to identify the CD5+NGC type of DLBCL as poor prognostic factor in DLBCL, and R-CHOP therapy could not improved their prognosis. The new treatment strategy should be planned as soon as possible, such as intensive chemotherapy or immunotherapy. DisclosuresMishima:Chugai Pharmaceutical, Inc: Other: consignment job. Terui:Novartis Pharma K.K.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Celgene Corporation: Speakers Bureau; Bristol-Myers Squibb company: Speakers Bureau. Yokoyama:Chugai Pharmaceutical, Inc: Other: consignment job. Nishimura:Chugai Pharmaceutical, Inc: Other: consignment job. Hatake:AbbVie, Gilead, Celgene, Solasia, Pfizer, Bristol-Myers Squibb, Janssen, Ghugai: Research Funding; Mundipharma K.K.: Honoraria.

Growth faltering regardless of chronic diarrhea is associated with mucosal immune dysfunction and microbial dysbiosis in the gut lumen

Despite the impact of childhood diarrhea on morbidity and mortality, our understanding of its sequelae has been significantly hampered by the lack of studies that examine samples across the entire intestinal tract. Infant rhesus macaques are naturally susceptible to human enteric pathogens and recapitulate the hallmarks of diarrheal disease such as intestinal inflammation and growth faltering. Here, we examined intestinal biopsies, lamina propria leukocytes, luminal contents, and fecal samples from healthy infants and those experiencing growth faltering with distant acute or chronic active diarrhea. We show that growth faltering in the presence or absence of active diarrhea is associated with a heightened systemic and mucosal pro-inflammatory state centered in the colon. Moreover, polyclonal stimulation of colonic lamina propria leukocytes resulted in a dampened cytokine response, indicative of immune exhaustion. We also detected a functional and taxonomic shift in the luminal microbiome across multiple gut sites including the migration of Streptococcus and Prevotella species between the small and large intestine, suggesting a decompartmentalization of gut microbial communities. Our studies provide valuable insight into the outcomes of diarrheal diseases and growth faltering not attainable in humans and lays the groundwork to test interventions in a controlled and reproducible setting.

Effects of Different Bone Substitutes on Reactive Oxygen Species Release in Leukocytes in Vitro: A Pilot Study.

To evaluate the formation of reactive oxygen species in human leukocytes promoted by bone substitutes that are different in origin and morphology used for jawbone tissue regeneration. This preclinical prospective randomized crossover study involved 10 subjects, from whom venous blood samples were taken. Leukocytes were separated and standardized. Sixty experimental samples consisted of leukocytes incubated with allogeneic, xenogeneic, or alloplastic bone substitutes at different bone weights (12.5 and 25 mg). The control samples consisted only of incubated leukocytes. Reactive oxygen species were quantitatively determined with the fluorimetric method. Statistical analysis was carried out using SPSS 23 software. The highest average reactive oxygen species values were obtained in the allogeneic bone substitute group (P < .05), while the xenogeneic bone substitute group and control group presented equal reactive oxygen species formation rates (P > .05). A proportional difference (P < .05) of reactive oxygen species emission was obtained between different masses of bone substitute in the samples. Allogeneic and alloplastic bone substitutes affect leukocytes and promote reactive oxygen species emission. Xenogeneic bone substitute presents no leukocyte stimulation and maintains anti-inflammatory conditions. Larger bone substitute mass provokes greater oxidative stress.

Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation.

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.

Characterization of the turbot Scophthalmus maximus (L.) myeloperoxidase. An insight into the evolution of vertebrate peroxidases

We have completed the characterization of the turbot (Scophthalmus maximus) myeloperoxidase (mpx) gene and protein, which we partially described in a previous study. The turbot mpx gene has 15 exons that encode a protein of 767 aa, with a signal peptide, propeptide and light and heavy chains, and also with haem cavities, a Ca+2-binding motif and several N- and O-glycosylation sites. The mature protein forms homodimers of about 150 kDa and is very abundant in turbot neutrophils. In addition to the mpx (epx2a) gene, another three peroxidase genes, named epx1, epx2b1 and epx2b2, were identified in the turbot genome. Epx1, Epx2b1 and Epx2b2 proteins also have signal peptides and many structural characteristics of mammalian MPO and eosinophil peroxidase (EPX). Mpx was strongly expressed in head kidney, while epx2b1 and epx2b2 were strongly expressed in the gills, and epx1 was not expressed in any of the tissues or organs analysed. In vitro stimulation of head kidney leucocytes with the parasite Philasterides dicentrarchi caused a decrease in mpx expression and an increase in epx2b1 expression over time. In turbot infected experimentally with P. dicentrarchi a significant increase in mpx expression in the head kidney was observed on day 7 postinfection, while the other genes were not regulated. However, mpx, epx2b1 and epx2b2 were downregulated in the gills of infected fish, and epx1 expression was not affected. These results suggest that the four genes responded differently to the same stimuli. Interestingly, BLAST analysis revealed that Epx1 and Mpx showed greater similarity to mammalian EPX than to MPO. Considering the phylogenetic and synteny data obtained, we concluded that the epx/mpx genes of Gnathostomes can be divided into three main clades: EPX1, which contains turbot epx1, EPX2, which contains turbot mpx (epx2a) and epx2b1 and epx2b2 genes, and a clade containing mammalian EPX and MPO (EPX/MPO). EPX/MPO and EPX2 clades share a common ancestor with the chondrichthyan elephant shark (Callorhinchus milii) and the coelacanth (Latimeria chalumnae) peroxidases. EPX2 was only found in fish and includes two sister groups. One of the groups includes turbot mpx and was only found in teleosts. Finally, the other group contains epx2b1 and epx2b2 genes, and epx2b1-2b2 loci share orthologous genes with other teleosts and also with holosteans, suggesting that these genes appeared earlier on than the mpx gene.