Healthy Human Blood Research Articles

Study the Effect of the Nanomaterial of the Gold Metal Prepared by Laser Ablation Method on Healthy Human Blood

The golden nanoparticles were prepared by pulse laser ablation (PLA) by the liquid phase method. Lasers(Nd: Yag) (1064nm (80mJ) wavelength) are used in different pulses (200, 400, and 600), and deionizeddistilled liquid water (DDDW) is used The prepared nanomaterial was added by laser ablation and withthree laser pulses to a peaceful blood sample, where the nanomaterial was added in different concentrations2, 4, 5 ml to 1 ml of blood. A blood image was taken for each sample, where the total samples became 9samples and compared with a sample of healthy blood. It was observed that the general observation of ratesand in general of all components of blood tissues has decreased significantly and very dangerous and alsofrightening with increasing concentration of both nanomaterial’s and increased number of laser pulses perminute compared to normal rates and control sample readings

Systemic and respiratory T-cells induced by seasonal H1N1 influenza protect against pandemic H2N2 in ferrets

Traditional influenza vaccines primarily induce a narrow antibody response that offers no protection against heterosubtypic infections. Murine studies have shown that T cells can protect against a broad range of influenza strains. However, ferrets are a more potent model for studying immune correlates of protection in influenza infection. We therefore set out to investigate the role of systemic and respiratory T cells in the protection against heterosubtypic influenza A infections in ferrets. H1N1-priming induced systemic and respiratory T cells that responded against pandemic H2N2 and correlated with reduced viral replication and disease. CD8-positive T cell responses in the upper and lower respiratory tract were exceptionally high. We additionally confirmed that H2N2-responsive T cells are present in healthy human blood donors. These findings underline the importance of the T cell response in influenza immunity and show that T cells are a potent target for future universal influenza vaccines.

5-Dodecanolide inhibits biofilm formation and virulence of Streptococcus pyogenes by suppressing core regulons of virulence

This study determined the antibiofilm and antivirulence potential of 5-Dodecanolide (DD) against an exclusive human pathogen Streptococcus pyogenes. Biofilm quantification assay showed antibiofilm efficacy of DD with maximum biofilm inhibition of 85% at 225 μg/mL concentration. Efficacy of antibacterial property of DD (225 μg/mL) was confirmed by CFU analysis and Alamar blue assay. Microscopic analyses evidently confirmed micro-colony formation, biofilm thickness and surface coverage were reduced upon DD treatment. In addition, based on the results of in vitro assays, it was noted that DD impaired the synthesis of surface hydrophobicity, slime, hyaluronic acid, hemolysin and protease production. Interestingly, DD increased the autoaggregation of S. pyogenes hence, facilitated enhanced recognition of clumped bacterial cells for innate immune clearance. The results were further validated by the reduced survival of DD treated S. pyogenes in healthy human blood. Consequently, based on the qPCR analysis DD altered the expression of core regulons srv, ropB, mga and genes associated with biofilm formation and virulence such as speB, dltA, srtB, sagA and slo. Hence, the overall results of the present study for the first time revealed the antibiofilm and antivirulence property of DD against clinically important pathogen S. pyogenes and further clinical investigations are required to assess the therapeutic use of DD.

Bioimprint Mediated Label-Free Isolation of Pancreatic Tumor Cells from a Healthy Peripheral Blood Cell Population.

New techniques are required for earlier diagnosis and response to treatment of pancreatic cancer. Here, a label-free approach is reported in which circulating pancreatic tumor cells are isolated from healthy peripheral blood cells via cell bioimprinting technology. The method involves pre-fabrication of pancreatic cell layers and sequential casting of cell surfaces with a series of custom-made resins to produce negative cell imprints. The imprint is functionalized with a combination of polymers to engineer weak attraction to the cells which is further amplified by the increased area of contact with the matching cells. A flow-through bioimprint chip is designed and tested for selectivity toward two pancreatic tumor cell lines, ASPC-1 and Mia-PaCa-2. Healthy human peripheral blood mononuclear cells (PBMCs) are spiked with pancreatic tumor cells at various concentrations. Bioimprints are designed for preferential retention of the matching pancreatic tumor cells and with respect to PBMCs. Tumor bioimprints are capable of capturing and concentrating pancreatic tumor cells from a mixed cell population with increased retention observed with the number of seedings. ASPC-1 bioimprints preferentially retain both types of pancreatic tumor cells. This technology could be relevant for the collection and interrogation of liquid biopsies, early detection, and relapse monitoring of pancreatic cancer patients.

Vaginal neutrophils eliminate sperm by trogocytosis.

What is the vaginal polymorphonuclear (PMN) spermicidal mechanism to reduce the excess of sperm? We show that PMNs are very efficient at killing sperm by a trogocytosis-dependent spermicidal activity independent of neutrophil extracellular traps (NETs). Trogocytosis has been described as an active membrane exchange between immune cells with a regulatory purpose. Recently, trogocytosis has been reported as a mechanism which PMNs use to kill tumour cells or Trichomonas vaginalis. We used in vivo murine models and human ex vivo sperm and PMNs to investigate the early PMN-sperm response. We set up a live/dead sperm detection system in the presence of PMNs to investigate in vivo and ex vivo PMN-spermicidal activity by confocal microscopy, flow cytometry and computer-assisted sperm analysis (SCA). We revealed that PMNs are highly efficient at killing sperm by way of a NETs-independent, contact-dependent and serine proteases-dependent engulfment mechanism. PMNs 'bite' sperm and quickly reduce sperm motility (within 5 min) and viability (within 20 min) after contact. N/A. This study was conducted using murine models and healthy human blood PMNs; whether it is relevant to human vaginal PMNs or to cases of infertility is unknown. Vaginal PMNs attack and immobilize excess sperm in the vagina by trogocytosis because sperm are exogenous and may carry pathogens. Furthermore, this mechanism of sperm regulation has low mucosal impact and avoids an exacerbated inflammatory response that could lead to mucosal damage or infertility. This work was partially supported by Ministry of Economy and Competitiveness ISCIII-FIS grants, PI16/00050, and PI19/00078, co-financed by ERDF (FEDER) Funds from the European Commission, 'A way of making Europe' and IiSGM intramural grant II-PI-MRC-2017. M.R. holds a Miguel Servet II contract (CPII14/00009). M.C.L. holds IiSGM intramural contract. There are no competing interests.

Teclistamab is an active T cell–redirecting bispecific antibody against B-cell maturation antigen for multiple myeloma

AbstractB-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiological conditions using healthy human whole blood, teclistamab mediated dose-dependent lysis of H929 cells and activation of T cells. Antitumor activity of teclistamab was also observed in 2 BCMA+ MM murine xenograft models inoculated with human T cells (tumor inhibition with H929 model and tumor regression with the RPMI 8226 model) compared with vehicle and antibody controls. The specific and potent activity of teclistamab against BCMA-expressing cells from MM cell lines, patient samples, and MM xenograft models warrant further evaluation of this bispecific antibody for the treatment of MM. Phase 1 clinical trials (monotherapy, #NCT03145181; combination therapy, #NCT04108195) are ongoing for patients with relapsed/refractory MM.

The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells.

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.

TLR engagement induces ARID3a in human blood hematopoietic progenitors and modulates IFNα production

The DNA binding protein AT-rich interacting domain 3a (ARID3a)22ARID3a, AT-rich interacting domain 3a is expressed in healthy human hematopoietic cord blood progenitors where its modulation influences myeloid versus B lineage development. ARID3a is also variably expressed in subsets of adult peripheral blood hematopoietic progenitors where the consequences of ARID3a expression are unknown. In B lymphocytes, Toll-like receptor (TLR)33TLR, Toll-like receptor signaling induces ARID3a expression in association with Type I interferon inflammatory cytokines. We hypothesized that TLR ligand stimulation of peripheral blood hematopoietic progenitors would induce ARID3a expression resulting in interferon production, and potentially influencing lineage decisions. Our data revealed that the TLR9 agonist CpG induces ARID3a expression with interferon alpha synthesis in human hematopoietic progenitors. However, ARID3a expression was not associated with increased B lineage development. These results demonstrate the need for further experiments to better define how pathogen-associated responses influence hematopoiesis.

Sequence-based prediction of SARS-CoV-2 vaccine targets using a mass spectrometry-based bioinformatics predictor identifies immunogenic T cell epitopes

BackgroundThe ongoing COVID-19 pandemic has created an urgency to identify novel vaccine targets for protective immunity against SARS-CoV-2. Early reports identify protective roles for both humoral and cell-mediated immunity for SARS-CoV-2.MethodsWe leveraged our bioinformatics binding prediction tools for human leukocyte antigen (HLA)-I and HLA-II alleles that were developed using mass spectrometry-based profiling of individual HLA-I and HLA-II alleles to predict peptide binding to diverse allele sets. We applied these binding predictors to viral genomes from the Coronaviridae family and specifically focused on T cell epitopes from SARS-CoV-2 proteins. We assayed a subset of these epitopes in a T cell induction assay for their ability to elicit CD8+ T cell responses.ResultsWe first validated HLA-I and HLA-II predictions on Coronaviridae family epitopes deposited in the Virus Pathogen Database and Analysis Resource (ViPR) database. We then utilized our HLA-I and HLA-II predictors to identify 11,897 HLA-I and 8046 HLA-II candidate peptides which were highly ranked for binding across 13 open reading frames (ORFs) of SARS-CoV-2. These peptides are predicted to provide over 99% allele coverage for the US, European, and Asian populations. From our SARS-CoV-2-predicted peptide-HLA-I allele pairs, 374 pairs identically matched what was previously reported in the ViPR database, originating from other coronaviruses with identical sequences. Of these pairs, 333 (89%) had a positive HLA binding assay result, reinforcing the validity of our predictions. We then demonstrated that a subset of these highly predicted epitopes were immunogenic based on their recognition by specific CD8+ T cells in healthy human donor peripheral blood mononuclear cells (PBMCs). Finally, we characterized the expression of SARS-CoV-2 proteins in virally infected cells to prioritize those which could be potential targets for T cell immunity.ConclusionsUsing our bioinformatics platform, we identify multiple putative epitopes that are potential targets for CD4+ and CD8+ T cells, whose HLA binding properties cover nearly the entire population. We also confirm that our binding predictors can predict epitopes eliciting CD8+ T cell responses from multiple SARS-CoV-2 proteins. Protein expression and population HLA allele coverage, combined with the ability to identify T cell epitopes, should be considered in SARS-CoV-2 vaccine design strategies and immune monitoring.

Characterization and origins of cell-free mitochondria in healthy murine and human blood

Intact cell-free mitochondria have been reported in microparticles (MPs) in murine and human bodily fluids under disease conditions. However, cellular origins of circulating extracellular mitochondria have not been characterized. We hypothesize that intact, cell-free mitochondria from heterogeneous cellular sources are present in the circulation under physiological conditions. To test this, circulating MPs were analyzed using flow cytometry and proteomics. Murine and human platelet-depleted plasma showed a cluster of MPs positive for the mitochondrial probe MitoTracker. Transgenic mice expressing mitochondrial-GFP showed GFP positivity in plasma MPs. Murine and human mitochondria-containing MPs were positive for the platelet marker CD41 and the endothelial cell marker CD144, while hematopoietic CD45 labeling was low. Both murine and human circulating cell-free mitochondria maintained a transmembrane potential. Circulating mitochondria were able to enter rho-zero cells, and were visualized using immunoelectron microscopic imaging. Proteomics analysis identified mitochondria specific and extracellular vesicle associated proteins in sorted circulating cell-free human mitochondria. Together the data provide multiple lines of evidence that intact and functional mitochondria originating from several cell types are present in the blood circulation.

The aging of P. falciparum infected RBCs by 2D-correlation Raman and EPR spectroscopy

Malaria is still a global health problem, especially when infection is caused by P. falciparum. In addition to its occurrence in endemic areas, it also takes the form of malaria imported by travelers. To understand the course of the disease, changes in the infected with P. falciparum red blood cells, in the process of their aging, were analyzed using Raman and EPR spectroscopy and the generalized 2D- COS method. The results were compared with the outcome of the natural aging process of human healthy blood cells. 2D correlation Raman synchronous peaks result from changes which reveals changes in erythrocytes caused by parasite invasion. First changes are connected with heme degradation- the aggregated heme and finally hemozoin. Other cross-peaks demonstrated the presence of hemoglobin breakdown products, which shows for example as the asparagine hemoglobinase impact. There are also observed complex changes in the erythrocyte membrane as the result of P. falciparum activity. The 2D asynchronous peaks are generated by phospholipids and the amino acids, His and Phe, that point to characteristic for P. falciparum protein family. The appearing 2D EPR correlation cross-peaks mainly come from iron ions, copper ions and peroxyl radicals. Iron ions in low- and high-spin complexes, related to the change of the geometry of paramagnetic centers, are the source of asynchronous cross-peaks indicating a complex process of iron transfer in the blood.

The effect of hyperoxia on inflammation and platelet responses in an ex vivo extracorporeal membrane oxygenation circuit.

Use of extracorporeal membrane oxygenation (ECMO) is expanding, however, it is still associated with significant morbidity and mortality. Activation of inflammatory and innate immune responses and hemostatic alterations contribute to complications. Hyperoxia may play a role in exacerbating these responses. Nine ex vivo ECMO circuits were tested using fresh healthy human whole blood, with two oxygen levels: 21% inspired fraction of oxygen (FiO2 ; mild hyperoxia; n=5) and 100% FiO2 (severe hyperoxia; n=4). Serial blood samples were taken for analysis of platelet aggregometry, leukocyte activation, inflammatory, and oxidative stress markers. ECMO resulted in reduced adenosine diphosphate- (P<.05) and thrombin receptor activating peptide-induced (P<.05) platelet aggregation, as well as increasing levels of the neutrophil activation marker, neutrophil elastase (P=.013). Additionally, levels of the inflammatory chemokine interleukin-8 were elevated (P<.05) and the activity of superoxide dismutase, a marker of oxidative stress, was increased (P=.002). Hyperoxia did not augment these responses, with no significant differences detected between mild and severe hyperoxia. Our ex vivo model of ECMO revealed that the circuit itself triggers a pro-inflammatory and oxidative stress response, however, exposure to supra-physiologic oxygen does not amplify that response. Extended-duration studies and inclusion of an endothelial component could be beneficial in characterizing longer term changes.

The role of neutrophil density in neutrophils-mediated inflammatory response induced by monosodium urate crystals

Objective: To study the role of neutrophil density and molecular mechanism in neutrophils-mediated inflammatory response induced by monosodium urate (MSU) crystals. Methods: Polymorphonuclear neutrophils (PMNs) isolated from healthy human peripheral blood were treated with MSU crystals at different density (5×10(6)/ml, 20×10(6)/ml, 100×10(6)/ml) in vitro. The mean fluorescence intensity (MFI) of PMNs and production of reactive oxygen species (ROS) were detected by flow cytometry. The distribution of MSU crystals was observed by polarized light microscopy. The neutrophil extracellular traps (NETs) formation was detected by immune fluorescence. The cytokines in cell supernatant were measured by beads assay including interleukin 1β (IL-1β) , tumor necrosis factor α (TNFα) , interleukin 8 (IL-8) , interferon inducible protein 10 (IP-10) , macrophage inflammatory protein 1 (MIP-1) , monokine induced by interferon-γ (MIG) , macrophage inflammatory protein 1α (MIP-1α) , macrophage inflammatory protein 1β (MIP-1β) . Results: (1) After MSU crystal intervention, the side scatters (SSC) of neutrophils with medium-cell density (20×10(6)/ml) and high-cell density (100×10(6)/ml) were 128±13 and 93±9 respectively, both significantly lower than 170±19 in low-cell density (5×10(6)/ml) group.(2) Similarly, compared with low-cell density group, the MFI (lucifer yellow) of PMNs with high-cell density was 1.8±0.2, also significantly decreased (P<0.05). When co-treated with oxygenated adenosine triphosphate (oxATP), MFI of PMNs were all enhanced consistently. (3) In MSU crystals stimulated PMNs, after adding 2',7'-dichlorodihydrofluorescein diacetate, the MFI values were 0.85±0.32, 2.49±0.78, 4.54±1.02 in low cell density groups, medium cell density groups, and high cell density groups respectively, indicating that the generation of ROS was positively correlated with the increase of PMN density (P<0.05). After the intervention of oxATP, the ROS production was significantly reduced. (4) MSU crystal induced NETs formation, especially at high cell density. NETs formation promotes MSU crystal aggregation, which could be partially overcome by oxATP pretreatment. (5) The expression of cytokines were all significantly decreased in the supernatant of PMNs at high cell density exposed to MSU crystals compared with PMNs at medium cell density (P<0.05) . Conclusion: The PMN-mediated inflammation induced by MSU crystals is cell density dependent, and ATP may play a role in partially overcoming the process.

Extracellular AMP Suppresses Endotoxemia-Induced Inflammation by Alleviating Neutrophil Activation

Intracellular adenosine monophosphate (AMP) is indispensable for cellular metabolic processes, and it is interconverted to ADP and/or ATP or activates AMP-activated protein kinase (AMPK). However, the specific biological function of extracellular AMP has not been identified. We evaluated the effect of extracellular AMP using in vivo and in vitro models of endotoxemia. We found that AMP inhibited inflammation and neutrophil activation in lipopolysaccharide (LPS)-induced endotoxemic mice. The effects of extracellular AMP were abolished by an adenosine 1 receptor (A1R) antagonist but were not influenced by inhibiting the conversion of AMP to adenosine (ADO), indicating that AMP inhibited inflammation by directly activating A1R. In addition, in vitro experiments using LPS-stimulated mouse neutrophils showed that AMP inhibited LPS-induced reactive oxygen species (ROS) production, degranulation, and cytokine production, while the effects were reversed by an A1R antagonist. Further research showed that AMP regulated LPS-stimulated neutrophil functions by inhibiting the p38 MAPK pathway. These findings were also confirmed in primary neutrophils derived from healthy human blood. Moreover, we collected serum samples from septic patients. We found that AMP levels were increased compared with those of healthy volunteers and that AMP levels were negatively correlated with disease severity. Together, these data provide evidence that extracellular AMP acts on A1R to suppress endotoxemia-induced inflammation by inhibiting neutrophil overactivation and that the p38 MAPK signaling pathway is involved.

Cirrhotic Endothelial Progenitor Cells Enhance Liver Angiogenesis and Fibrosis and Aggravate Portal Hypertension in Bile Duct-Ligated Cirrhotic Rats.

BackgroundCirculating cirrhotic endothelial progenitor cells (EPC) interact with both liver sinusoidal endothelial cells (LSEC) and hepatic stellate cells (HSC) and promote angiogenesis in vitro. This study evaluated the effect of cirrhotic and control EPCs on hepatic angiogenesis, microcirculation, and fibrosis in vivo in rat models of cirrhosis.MethodologyAnimal models of cirrhosis were prepared by bile duct ligation (BDL). Circulating EPCs isolated from healthy human and cirrhotic blood were characterized by flow cytometry, cultured and administered through the tail vein in BDL rats after 2 weeks of ligation. The cells were given thrice a week for 2 weeks. The untreated group of BDL rats received only saline. Fibrosis was evaluated by Masson’s trichrome staining. Dedifferentiated LSECs were identified by the expression of CD31, and activated HSCs were marked as alpha-SMA-positive cells and were studied by immunohistochemistry and western blotting in saline-, healthy EPC-, and cirrhotic EPC-treated rats. In vivo, hepatic and systemic hemodynamic parameters were evaluated. Liver functions were evaluated.ResultsIn comparison to controls, BDL rats revealed an increase of fibrosis and angiogenesis. Among the treated rats, cirrhotic EPC-treated rats had increased fibrosis grade as compared to healthy EPC-treated and saline-treated rats. There was an increase of both fibrosis and angiogenesis markers, alpha-SMA and CD31 in cirrhotic EPC-treated rats as compared to healthy EPC-treated and saline-treated rats in immunohistochemistry and western blot studies. Cirrhotic EPC-treated BDL rats had high portal pressure and portal blood flow with significantly elevated hepatic vascular resistance in comparison with healthy EPC- and saline-treated BDL animals, without significant differences in mean arterial pressure. Cirrhotic EPC-treated BDL rats also showed a substantial increase in the hepatic expression of angiogenic receptors, VEGFR2 and CXCR4 in comparison with saline-treated rats.ConclusionThe study suggests that transplantation of cirrhotic EPCs enhances LSEC differentiation and angiogenesis, activates HSCs and worsens fibrosis, thus resulting in hepatic hemodynamic derangements in BDL-induced cirrhosis.

Effect of cell geometry in the evaluation of erythrocyte viscoelastic properties.

The red blood cell membrane-cytoskeleton is a complex structure mainly responsible for giving the cell rigidity and shape. It also provides the erythrocyte with the ability to pass through narrow capillaries of the vertebrate blood circulatory system. Although the red blood cell viscoelastic properties have been extensively studied, reported experimental data differ by up to three orders of magnitude. This could be attributed to the natural cell variability, to the different techniques employed, and also to the models used for the cell response, which are highly dependent on cell geometry. Here, we use two methodologies based on optical tweezers to investigate the viscoelastic behavior of healthy human red blood cells, one applying small cell deformations (microrheology) and another imposing large deformations (tether extraction). We also establish a defocusing microscopy-based method to characterize the cell geometry and thus the erythrocyte form factor, an essential parameter that allows comparisons among the viscoelastic properties at different conditions. Moreover, for small deformations, a soft glassy rheology model is used to discuss the results, while for large deformations two surface shear moduli and one surface viscosity are determined, together with the surface tension and bending modulus of the erythrocyte membrane lipid component. We also show that F-actin is not detected in tethers, although the erythrocyte membrane has physical properties like those of other adherent cells, known to have tethers containing F-actin inside. Altogether, our results show good agreement with the reported literature and we argue that, to properly compare the viscoelastic properties of red blood cells in different situations, the task of cell geometry characterization must be accomplished. This may be especially important when the influence of agents, like the malaria parasite, induces changes in both the geometry and chemical constituents of the erythrocyte membrane. Together, the new methodologies and procedures used in this study would allow the erythrocyte community to better explore the mechanical behavior of red blood cells and may be useful to characterize erythrocyte viscoelasticity changes in several blood diseases.

Comparison of rigid polymer vials and flexible bags for cryopreservation of T cells

Background & Aim The commercialization of cells for therapeutic use drives a critical need for the refinement of storage and handling procedures to ensure the integrity of cell-based products is maintained from manufacture through until administration to the patient. Autologous cell therapy applications currently call for large volume cell suspensions administered via intravenous injection, making cryogenic flexible bags an attractive containment solution. However, numerous challenges exist when using bags made from materials such as ethylene vinyl acetate (EVA) including filling difficulties, cell product loss due to dead volume, and cracking of the flexible material during the freeze/thaw process. Cyclic olefin polymer (COP) vials with rubber stopper-aluminum seal closures have desirable container closure integrity and material properties for cell therapy applications and have been shown to be compatible with numerous cell types in small volume (2 mL) format. This work evaluates the performance of larger 50 mL COP vials as an alternative for cryogenic storage of T cells. Methods, Results & Conclusion Peripheral blood mononuclear cells were isolated from healthy human donor blood and T cells were subsequently selected, stimulated, and expanded for 7 days. The T cells were then placed into either flexible EVA bags or rigid COP vials to be frozen for storage using a well-controlled process. The containers were thawed and the cells were cultured for a further 7 days. Cell growth, cell viability, T cell subsets, and exhaustion markers were evaluated between the first expansion and freezing, to establish the baseline. Container cryopreservation performance was determined by evaluating the same readouts immediately post-thaw and after the second expansion. Results indicate that the freezing and expansion processes may affect T cell subsets and exhaustion. The T cell subsets, exhaustion, and all other readouts examined revealed comparable results whether the cells were stored in a flexible bag or a rigid vial despite differences in cross-sectional area and time required to thaw. The findings highlight the value of understanding a cell-based product in the context of its handling and storage. Furthermore, the study has shown that 50 mL vials can provide a viable alternative for final containment of cell suspensions at low-temperature.

Neuropilin-1 Amplifies Regulatory T cell Function in Human Cancer

Abstract Regulatory T cells (Tregs) maintain peripheral tolerance but also contribute to cancer progression by dampening anti-tumor immunity. Knocking out neuropilin-1 (NRP1) on murine Tregs attenuates tumor growth by rendering intratumoral Tregs non-suppressive. Whereas NRP1 is constitutively expressed on all mouse Tregs, NRP1 is infrequently expressed on Tregs in the peripheral blood of healthy humans. We hypothesized that (1) NRP1 expression is upregulated in cancer patients, (2) NRP1 marks highly suppressive human Tregs, and (3) sustained Treg activation initiates NRP1 expression. NRP1+ Tregs are enriched in tumors across numerous cancer types but largely devoid in non-cancerous inflamed and healthy tissues. Intratumoral NRP1 expression negatively correlates with disease-free survival in head and neck squamous cell carcinoma (HNSCC; p = 0.03; Hazard ratio = 5.4). Intratumoral NRP1+ Tregs upregulate a suppressive module indicated by co-expression of TIGIT, CCR8, and multiple TNF receptor super family members. Correspondingly, NRP1+ Tregs potently suppress CD8+ T cell proliferation in vitro, which can be disrupted with NRP1-specific blockade. Furthermore, Treg activation in vitro drives NRP1 expression, and sustained TCR signals are required to maintain NRP1 expression. We conclude that NRP1+ Tregs are selectively enriched in human cancer and oppose anti-tumor immunity. Therefore, destabilizing intratumoral Tregs using NRP1 blockade may complement other T cell therapies such as anti-PD1 blockade.

Identification of N-formylated peptides as the neutrophilic chemotactic factors of Mycobacterium tuberculosis

Abstract Since tuberculosis (TB) remains a major cause of human death, it is urgent to understand the pathology of TB. Lung neutrophil infiltration initiates granuloma formation and is associated with disease severity. Though several cytokines and chemokines are associated with neutrophil infiltration in chronic inflammation, neutrophil chemotactic factors of Mycobacterium tuberculosis (Mtb) remain unexplored. Thus, we performed neutrophil chemotactic assays by culturing neutrophils of healthy human blood samples and mouse bone marrow in the upper chamber of a transwell plate with 5–8 μm pores with the culture filtrates of Mtb H37Rv (CF) in the lower chamber using N-formyl-met-leu-phe (fMLF), a bacterial neutrophil chemotactic tripeptide, as a positive control. The cell numbers in the lower chamber were determined by flow cytometry as a chemotactic indicator. Our results show that CF induced chemotaxis of both human and mouse neutrophils in an Mtb culture period dependent manner. Testing CF of 11 clinical isolates of Mtb also showed neutrophil chemotactic activity. Sulfasalazine, a formylated peptide receptor inhibitor, blocked chemotaxis of neutrophils, indicating the involvement of fPRs in CF induces chemotaxis of neutrophils. Mass spectrometry analysis of CF identified three candidate N-formylated heptapeptides. Authentication of the chemotactic activities of the identified peptides with synthetic mimetics confirmed their neutrophil chemotactic activities and indicated the requirement of N-formylation for their chemotactic activities. Thus, we have identified novel formylated peptides of Mtb with neutrophil chemotactic activities, and further studies are in progress to determine their significance in TB infection.

Inhibition of biofilm and biofilm-associated virulence factor production in methicillin-resistant Staphylococcus aureus by docosanol

Antimicrobial resistance is a major public health concern in infection control. Hence, a multi-pronged approach is necessary to curb the severity of infections. The present study entails the identification of docosanol (fatty alcohol) from Streptomyces as a novel antibiofilm agent which can target the virulence factors of MRSA. Results showed that docosanol as a potent antibiofilm agent and found to inhibit several virulence factors of MRSA. The antibiofilm efficacy of docosanol analyzed through light and scanning electron microscopy showed a significant reduction in adherent cells. Moreover, analysis of three-dimensional structure of biofilm matrix by confocal laser scanning microscope demonstrated effective antibiofilm potential of docosanol. In addition, docosanol reduced the survival rate of MRSA in healthy human blood and enhanced the neutrophil-mediated killing by interfering with hemolysin production. RT-qPCR analysis revealed the down regulation of several virulence genes, possibly by affecting the expression of the accessory gene regulator (agr) system and transcriptional regulator sarA. These findings suggest that docosanol could effectively reduce the biofilm phenotype and virulence production, and thus becomes a promising candidate to treat MRSA infections.