Isolation Of Peripheral Blood Mononuclear Cells Research Articles

Protocol for investigating intracellular microbial diversity using single-cell RNA-seq in immune cells of SARS-CoV-2-positive and recovered patients.

Intracellular microorganisms like viruses and bacteria impact immune cell function. However, detection of these microbes is challenging as the majority exist in a non-culturable state. This protocol presents detailed steps to investigate intracellular microbial diversity using single-cell RNA sequencing (scRNA-seq) in immune-cells of SARS-CoV-2-positive and recovered patients. We present a workflow from sample collection to library preparation, covering peripheral blood mononuclear cell (PBMC) isolation, single-cell labeling, cartridge priming, and cell lysis. We outline the steps for analyzing the scRNA-seq data, from data quality control (QC) to detection of intracellular microbes. For complete details on the use and execution of this protocol, please refer to Yadav etal.1.

Inactivated Toxoplasma gondii nanovaccine boosts T-cell memory response in a seropositive yellow-footed rock wallaby (Petrogale xanthopus) – A case report from Copenhagen Zoo

Toxoplasma gondii is a ubiquitous parasite causing significant mortality in captive wildlife, especially marsupials. Historically, treatment has been unrewarding and no vaccine was available. An intranasal vaccine based on purified inactivated T. gondii was developed for toxoplasmosis prevention. A vaccination campaign started in early 2017 and was successful in preventing toxoplasma-related mortality in marsupials in many European and South American zoos. Amongst the vaccinated wallabies, about 30% were T. gondii seropositive before the vaccination, and no toxoplasma-related deaths were observed since the administration of the vaccine. The objective of this case study was to assess the potential effect of the vaccination on a seropositive wallaby. It is important to note that this vaccine doesn't induce any humoral response in sheep, and squirrel monkeys but induces a strong T-cell response. A T. gondii seropositive Yellow-footed rock wallaby (Petrogale xanthopus) from Copenhagen Zoo received two doses of the aforementioned intranasal vaccine. Blood samples were collected before each vaccination and used for peripheral blood mononuclear cell isolation. The impact of the vaccination on the lymphocyte phenotype was characterized by flow cytometry. Cell size, represented by forward scatter, and granularity, represented by side scatter parameters were analyzed. Two doses of the vaccine induced a respective 30.1 and 25.6% increase in cell size and granularity in lymphocytes stimulated with T. gondii antigens, as assessed by flow cytometry. These changes were likely correlated with T-cell activation, which indicates that two doses of the vaccine might have boosted the already-existing T-cell memory response against T. gondii in a seropositive animal. No morphological changes were observed in lymphocytes from an unvaccinated seronegative wallaby. This is the first documented case of boosting an already-existing cellular immune response against toxoplasmosis by the vaccine in a seropositive Yellow-footed rock wallaby.

Peripheral blood mononuclear cells isolation from mouse and rabbit blood to quantify active nucleotide and define brincidofovir dose for smallpox.

Aim: Brincidofovir has been approved by the US FDA for the treatment of smallpox via the "Animal Rule". The active moiety, cidofovir diphosphate (CDV-PP), was measurable in human peripheral blood mononuclear cells (PBMCs), but quantitation in animals was challenging given their limited blood volume. The aim of this study was to optimize PBMC isolation from rabbit and mouse blood to allow quantitation of CDV-PP.Materials & methods: PBMC isolation methods using various separation media were evaluated using blood from naive and brincidofovir-dosed animals. CDV-PP was quantified using liquid chromatography tandem mass spectrometry.Results & conclusion: PBMC isolation yields were increased by >fourfold compared with yields obtained using human methods, allowing cross-species exposure comparisons.

Correlation of the treatment sensitivity of patient-derived organoids with treatment outcomes in patients with head and neck cancer (SOTO): protocol for a prospective observational study

IntroductionOrganoids have been successfully used in several areas of cancer research and large living biobanks of patient-derived organoids (PDOs) have been developed from various malignancies. The characteristics of the original...

Cryo-PRO facilitates whole blood cryopreservation for single-cell RNA sequencing of immune cells from clinical samples.

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated viadensity gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

The Effect of Subclinical Ketosis on the Peripheral Blood Mononuclear Cell Inflammatory Response and Its Crosstalk with Depot-Specific Preadipocyte Function in Dairy Cows.

During the periparturient period, cows undergo heightened energy demands at lactation onset, paired with reduced dry matter intake, leading to negative energy balance (NEB). Excessive lipolysis-driven adipose tissue remodeling, triggered by NEB, significantly contributes to ketosis in periparturient dairy cows. However, the role of peripheral blood mononuclear cells (PBMCs) in the pathogenesis of ketosis and in modulating adipose tissue function remains poorly understood. Here, we investigated how ketosis affects the transcriptional profile and secretome of PBMCs and its influence on preadipocyte function in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). Twenty-one postpartum Holstein dairy cows were categorized as either subclinical ketosis (SCK; BHB ≥ 1.0 mM) or control (CON; BHB < 0.8 mM) based on blood beta-hydroxybutyrate (BHB) concentration screening. Blood samples were collected intravenously for the isolation of PBMCs and serum metabolic profiling. Ketosis elevated circulating NEFA and BHB levels but reduced total WBC and neutrophil counts. Isolated PBMCs were evaluated for gene expression and used to produce conditioned media (PBMC-CM), during which PBMCs were stimulated with 10 ng/mL LPS. The overall phenotype of PBMCs was largely consistent between SCK and CON cows, with minimal differences detected in immunomodulatory cytokine expression and PBMC-CM composition following stimulation. Preadipocytes isolated from non-ketotic cows were treated with PBMC-CM to assess the effect of PBMC secretomes on adipose cell function. Preadipocytes treated with SCK PBMC-CM showed reduced lipid accumulation compared to those treated with CON PBMC-CM regardless of the depot. SAT preadipocytes had heightened expression of lipid metabolism-related genes, including DGAT1, LIPE, and FASN, compared to VAT when treated with SCK PBMC-CM. Preadipocytes treated with CM from PBMC stimulated by LPS exhibited upregulation in IL1B and IL6 regardless of the depot or source of PBMCs. Together, these results indicate that although PBMC profiles showed minimal differences, preadipocytes treated with PBMC-CM may be influenced by additional factors, leading to altered preadipocyte function and gene expression that may contribute to adipose cellular dysfunction.

Expression of JAK/STAT Signaling Proteins at Diagnosis and Remission in Patients with Acute Myeloid Leukemia.

Acute myeloid leukemia (AML), among other malignancies, has been linked to the deregulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway, which is essential for cell growth, proliferation, and differentiation. This study aimed to investigate the expression of JAK/STAT proteins at diagnosis and remission and how it affects overall survival (OS). This is a prospective study conducted in the College of Medicine, Sultan Qaboos University. Using the enzyme-linked immunosorbent assay assay, we estimated the expression levels of JAK/STAT pathway proteins, including pJAK2, pJAK1, JAK1, pSTAT3, pSTAT5, and STAT5, in peripheral blood and bone marrow samples at diagnosis and remission and normalized to the total protein content. Twenty-three adult AML patients (median age = 47) and seven healthy volunteers, who were used as controls, were included in the study. A total of 68 cell lysates were extracted from samples using peripheral blood mononuclear cells isolation assay. OS was estimated using the Kaplan-Meier curve, and groups were compared with the COX regression model. The overexpression percent was calculated using the chi-square test (p > 0.050). The study revealed that the expression of JAK1, pJAK1, pJAK2, pSTAT3, STAT5, and pSTAT5 in peripheral blood was lower during the diagnosis phase compared to the remission phase (p > 0.050). All these proteins were overexpressed at diagnosis. These proteins did not impact the OS of AML. The expression levels of these proteins at the time of diagnosis do not significantly influence the OS of patients with AML. The study is limited in sample size and needs to be confirmed in future studies with a larger sample size.

Landscape and prognostic values of lymphocytes in patients with hepatocellular carcinoma undergoing transarterial embolization.

Transarterial embolization (TACE), the first-line treatment for hepatocellular carcinoma (HCC), does not always lead to promising outcomes in all patients. A better understanding of how the immune lymphocytes changes after TACE might be the key to improve the efficacy of TACE. However, there are few studies evaluating immune lymphocytes in TACE patients. Therefore, we aimed to evaluate the short- and long-term effects of TACE on lymphocyte subsets in patients with HCC to identify those that predict TACE prognosis. Peripheral blood samples were collected from 44 HCC patients at the following time points: one day before the initial TACE, three days after the initial TACE, and one month after the initial TACE and subjected to peripheral blood mononuclear cell isolation and flow cytometry. Dynamic changes in 75 lymphocyte subsets were recorded and their absolute counts were calculated. Tumor assessments were made every 4-6 weeks via computed tomography or magnetic resonance imaging. Our results revealed that almost all lymphocyte subsets fluctuated three days after TACE, but only Tfh and B cells decreased one month after TACE. Univariate and multivariate Cox regression showed that high levels of Th2 and conventional killer Vδ2 cells were associated with longer progressive-free survival (PFS) after TACE. Longer overall survival (OS) after TACE was associated with high levels of Th17 and viral infection-specific Vδ1 cells, and low levels of immature NK cells. In conclusion, TACE has a dynamic influence on the status of lymphocytes. Accordingly, several lymphocyte subsets can be used as prognostic markers for TACE.

Impaired autophagy following ex vivo cooling of simulated hypothermic temperatures in peripheral blood mononuclear cells from young and older adults

Hypothermia is a critical consequence of extreme cold exposure that increases the risk of cold-related injury and death in humans. While the initiation of cytoprotective mechanisms including the process of autophagy and the heat shock response (HSR) is crucial to cellular survival during periods of stress, age-related decrements in these systems may underlie cold-induced cellular vulnerability in older adults. Moreover, whether potential sex-related differences in autophagic regulation influence the human cold stress response remain unknown. We evaluated the effect of age and sex on mechanisms of cytoprotection (autophagy and the HSR) and cellular stress (apoptotic signaling and the acute inflammatory response) during ex vivo hypothermic cooling. Venous blood samples from 20 healthy young (10 females; mean [SD]: 22 [2] years) and 20 healthy older (10 females; 66 [5] years) adults were either isolated immediately (baseline) for peripheral blood mononuclear cells (PBMCs) or exposed to water bath temperatures maintained at 37, 35, 33, 31, or 4 °C for 90 min before PBMC isolation. Proteins associated with autophagy, apoptosis, the HSR, and inflammation were analyzed via Western blotting. Indicators of autophagic initiation and signaling (LC3, ULK1, and beclin-2) and the HSR (HSP90 and HSP70) increased when exposed to hypothermic temperatures in young and older adults (all p ≤ 0.007). Sex-related differences were only observed with autophagic initiation (ULK1; p = 0.015). However, despite increases in autophagic initiators ULK1 and beclin-2 (all p < 0.001), this was paralleled by autophagic dysfunction (increased p62) in all groups (all p < 0.001). Further, apoptotic (cleaved-caspase-3) and inflammatory (IL-6 and TNF-α) signaling increased in all groups (all p < 0.001). We demonstrated that exposure to hypothermic conditions is associated with autophagic dysfunction, irrespective of age or sex, although there may exist innate sex-related differences in cytoprotection in response to cold exposure as evidenced through altered autophagic initiation.

Evaluation of expression levels of microRNA processing elements in patients with sudden sensorineural hearing loss

BackgroundMicroRNAs have a significant role in the function and development of the hearing system. Idiopathic sudden sensorineural hearing loss (SSNHL) is a complicated disorder with no long-established reason. Since microRNAs play imperative roles in every aspect of the neural system, their dysregulation may contribute to the onset of SSNHL. The current study aimed to assess the expression patterns of microRNA processing elements (DROSHA, DICER, and DGCR-8) as the vital factors in microRNA biology that can affect the expression levels of microRNA. This study assessed DROSHA, DICER, and DGCR-8 mRNA expression levels in the peripheral blood mononuclear cells (PBMC) of 50 patients with SSNHL and 50 matched controls. After the isolation of PBMC, total RNA was extracted, and the expression levels of DROSHA, DICER, and DGCR-8 genes were evaluated using quantitative real-time PCR.ResultsThe results illustrated significant up-regulation of DICER and DGCR-8 genes in SSNHL patients at the mRNA level. Furthermore, despite no significant change in DROSHA level, DICER and DGCR-8 were significantly correlated with SSNHL. However, there was no significant correlation between these gene expressions and the clinicopathological features of patients.ConclusionThis study verified for the first time that the DGCR_8 and DICER mRNA expression levels were significantly up-regulated in patients with SSNHL, proposing that microRNAs and their processing pathways play key roles in the progression and development of SSNHL.

Abstract A078: In vivo tumor micro-environment characterization: preliminary results of an ovarian cancer patients-derived co-colture organoids platform

Abstract Introduction: Most patients with ovarian cancer (OC) are diagnosed at advanced stage. Current standard of care is based on surgery and platinum-based chemotherapy (CHT) in association with target therapies according to molecular characteristics (BRCA status and Homologous Recombination Repair (HRR) status). However, 15-20% progressed to first line CHT and mechanisms of resistance remain far from being completely known. In this scenario the role of tumoral microenvironment (TME) represents an open question. Methods: We designed a study to establish OC patients-derived co-colture organoids (ID PICTURE, IRB approval 4531). Both peripheral Blood Mononuclear Cells (PBMC) and isolated immune infiltrating cells from tumor tissue were planned to be used. The first endpoint of the study was the feasibility of this approach including (i) establishment OC patients-derived organoids (ii) PBMC isolation from whole blood and characterization (iii) immune infiltrate isolation from tumor tissue and characterization. For the latter, the evaluation of the immune phenotype was carried out by cytofluorimetry analyses, through target cells labelling with monoclonal antibodies conjugates with fluorescent molecules (CD45, CD19, CD3, CD4, CD8, 7AAD). Correlation between tumor immune infiltrate and mutational and clinical findings was also accomplished. Results: From January 2022 to May 2023, 36 patients with EOC, 92% with high grade serous OC and 95% with an advanced stage (from stage III according to FIGO, all high tumor load according to the Fagotti’s score) were enrolled. 23 patients (64%) were BRCA wild type while HRR status was available only for 17 patients of whom 28% were deficient. Considering the model feasibility, 5 organoids were obtained, and 8 and 20 million immune cells were isolated from tissue and peripheral blood, respectively. The immune infiltrate analysis was performed on 21 samples obtained from primary debulking surgery and 15 from diagnostic laparoscopies. 75% were treated with carboplatin paclitaxel (weekly or q21); among patients undergoing neoadjuvant treatment, 3 had a complete response (CRS3). The infiltration of B and T lymphocytes was described in all patients: 73% were positive for CD3, 20% for CD45, 7% for CD19. The lymphocytes T characterization was available for 7 patients with a median value of CD4+ 51.3, CD8+ 44.4, CD8+CD4+ 2,6 and CD8-CD4- 1.6. A trend for statistically significant difference was observed for CD45+ in stage IV vs stage I-III (p=0.021) and in CD19+ B cells in BRCA mutant vs BRCA WT (p=0.026). Finally, CD3+ T cells showed a significantly higher concentration in the 4 patients experiencing progression of disease at 6 months follow-up (p=0.014). Conclusion: These preliminary results suggest that patients-derived organoids co-cultured with tumor immune infiltrate could be challenging because of small numbers of overall cell content. Nevertheless, a correlation between specific components of B and T cells and molecular features and cancer progression deserves further investigation. Citation Format: Floriana Camarda, Clara Di Mario, Camilla Nero, Barbara Tolusso, Stefano Alivernini, Francesca Sillano, Sonia Mastrogiovanni, Tina Pasciuto, Elisa Gremese, Eleonora Cesari, Alessandra Ciucci, Claudio Sette, Giovanni Scambia. In vivo tumor micro-environment characterization: preliminary results of an ovarian cancer patients-derived co-colture organoids platform [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A078.

Alterations in mitochondria isolated from peripheral blood mononuclear cells and tumors of patients with epithelial ovarian cancers

Metabolic alterations play an essential role in ovarian carcinogenesis. The flexibility of mitochondrial functions facilitates cellular adaptation to the tough environment associated with carcinogenesis. An understanding of the differences in mitochondrial functions in normal ovaries and cancers could provide a basis for further exploration of future mitochondria-based screening, diagnosis, prognostic prediction, and targeted therapy for epithelial ovarian cancers. The main objective of this study was to assess mitochondrial function profiles measured from PBMCs and ovarian tissues of epithelial ovarian cancers in comparison with normal ovaries. A total of 36 patients were recruited for the study, all of whom underwent primary surgical treatment for malignant epithelial ovarian neoplasm. Of these, 20 patients were in the early stage and 16 patients were in the advanced stage. Additionally, 21 patients who had pelvic surgery for benign gynecologic conditions, with normal ovaries incidentally removed, were recruited as controls. At the time of surgery, a blood sample was collected from each participant for PBMC isolation, and ovarian tissue was retained for molecular studies. These studies included the examination of oxidative stress, mitochondrial mass, mitochondrial respiration, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP) changes, and mitochondrial swelling. Clinical and histopathological data were also collected and compared between different stages of epithelial ovarian cancers: early-stage (group 1), advanced-stage (group 2), and normal ovaries (group 3). The levels of cellular oxidative stress, mitochondrial mass, and mitochondrial biogenesis in the peripheral blood mononuclear cells (PBMCs) of participants with ovarian cancer were significantly lower than those of the control group. However, the mitochondrial respiratory parameters measured from the PBMCs were similar across all three groups. Furthermore, mitochondrial membrane depolarization and mitochondrial swelling were observed in ovarian tissues of both early-stage and advanced-stage cancer groups. We demonstrated the dynamic nature of mitochondrial ROS production, biogenesis, and respiratory function in response to epithelial ovarian carcinogenesis. The flexibility of mitochondrial functions under diverse conditions may make it a challenging therapeutic target for ovarian cancer.

Analysis of Human Peripheral Blood Mononuclear Cells in Patients with Neurodegenerative Diseases.

The role of immune system in the progression of neurodegenerative diseases has been studied for decades in animal models. However, invasive studies in human subjects remain controversial due to the heterogeneity of the presentation of different diagnostic categories at different stages of the disease. Peripheral blood mononuclear cells (PBMCs) contain immune cells including dendritic cells (DCs), monocytes, macrophages, and T lymphocytes. Isolating PBMCs from whole blood samples collected from patients provides a minimally invasive method for analyzing the immune system's function in patients with neurodegenerative diseases. By isolating single cell types from patients' peripheral blood, in vitro analyses can be conducted including RNA sequencing, immunofluorescence, and phagocytic analysis. In this chapter, we discuss PBMC separation and isolation of macrophages in pure culture in vitro. We also outline methods for performing RNA-seq on cultured macrophages and other techniques for investigating the role of macrophages in neurodegenerative disease pathophysiology.

Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus).

Elephants are susceptible to Mycobacterium tuberculosis (M. tb) complex (MTBC) infections. Diagnosis of tuberculosis (TB) in elephants is difficult, and most approaches used for human TB diagnosis are not applicable. An interferon gamma release assay (IGRA) to diagnose TB in Asian elephants (Elephas maximus) using peripheral blood mononuclear cells (PBMCs) has been previously developed. Although the assay is shown to be valid in determining MTBC infection status, the laborious PBMC isolation process makes it difficult to use. In this study, we simplified the method by using whole blood cultures (WC) as the starting material. Using PBMC cultures for IGRA, the MTBC infection status of 15 elephants was first confirmed. Among these animals, one has been previously confirmed for M. tb infection by both TB culture and PCR and the other was confirmed for MTBC infection in this study by droplet digital PCR (ddPCR) method. WC for IGRA consisted of an unstimulated sample, a mitogen stimulated sample, and sample stimulated with recombinant M. tb antigens, ESAT6 and CFP10. Using WC for IGRA in the 15 enrolled elephants, the results showed that 7 out of 15 samples yielded MTBC infection positive status that were completely concordant with those from the results using PBMCs. To test this method, WC for IGRA were applied in another elephant cohort of 9 elephants. The results from this cohort revealed a perfect match between the results from PBMC and WC. Responses to ESAT6 or CFP10 by PBMC and WC were not completely concordant, arguing for the use of at least two M. tb antigens for stimulation. Given the ease of sample handling, smaller blood sample volumes and equivalent efficacy relative to the PBMC approach, using WC for IGRA provides a novel, rapid, and user-friendly TB diagnostic method for determining the MTBC infection in elephants.

Effect of wash media type during PBMC isolation on downstream characterization of SARS-CoV-2-specific T cells

Protocols for the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood vary greatly between laboratories, especially in published studies of SARS-CoV-2-specific T cell responses following infection and vaccination. Research on the effects of different wash media types or centrifugation speeds and brake usage during the PBMC isolation process on downstream T cell activation and functionality is limited. Blood samples from 26 COVID-19-vaccinated participants were processed with different PBMC isolation methods using either PBS or RPMI as the wash media with high centrifugation speed and brakes or RPMI as the wash media with low speed and brakes (RPMI+ method). SARS-CoV-2 spike-specific T cells were quantified and characterized via a flow cytometry-based activation induced markers (AIM) assay and an interferon-γ (IFNγ) FluoroSpot assay and responses were compared between processing methods. Samples washed with RPMI showed higher AIM+ CD4 T cell responses than those washed with PBS and showed a shift away from naïve and towards an effector memory phenotype. The activation marker OX40 showed higher SARS-CoV-2 spike-induced upregulation on RPMI-washed CD4 T cells, while differences in CD137 upregulation were minimal between processing methods. The magnitude of the AIM+ CD8 T cell response was similar between processing methods but showed higher stimulation indices. Background frequencies of CD69+ CD8 T cells were increased in PBS-washed samples and were associated with higher baseline numbers of IFNγ-producing cells in the FluoroSpot assay. Slower braking in the RPMI+ method did not improve detection of SARS-CoV-2-specific T cells and caused longer processing times. Thus, the use of RPMI media with full centrifugation brakes during the wash steps of PBMC isolation was found to be most effective and efficient. Further studies are needed to elucidate the pathways involved in RPMI-mediated preservation of downstream T cell activity.

Gene expression of pregnancy-associated glycoproteins-1 (PAG-1), interferon-tau (IFNt) and interferon stimulated genes (ISGs) as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows

The aim of this study was to determine the presence of Pregnancy-associated glycoprotein −1 (PAG-1) mRNA expression in the maternal circulation of pregnant buffaloes during the early stage of pregnancy. Contemporaneously, the mRNA expression levels of Interferon-tau (IFNt) and some Interferon stimulated genes (ISGs) (interferon stimulated gene 15 ubiquitin-like modifier interferon, ISG15; Mixoviruses resistance 1 and 2, MX1 and MX2; 2′,5′-oligoadenylate synthase 1,OAS1) were evaluated in order to expand our knowledge of the molecular processes involved in the early stages of pregnancy and to identify potential biomarkers of maternal-fetal cellular interaction in buffalo. The study was conducted on 38 synchronized and artificially inseminated buffalo cows (d 0), divided ex post into 3 groups: Pregnant (n = 17), Non-pregnant (n = 15) and Embryo mortality (n = 6). Blood samples were collected on d 14, 19, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) isolation. Expression levels of mRNA of PAG-1, IFNt, ISG15. MX1, MX2 and OAS1 were measured using RT-qPCR. No significant changes were observed in IFNt and PAG gene expressions between groups, while significant differences (p < 0.001) were found for ISG15, MX1, MX2, and OAS1. Pairwise comparisons revealed that the differences between groups occurred on days 19 and 28 post-AI. ISG15 proved to have the best diagnostic performance for distinguishing between pregnant animals and animals that experienced embryo mortality with the ROC analysis. According to the results of the univariate analyses, day 19 was identified as the most indicative to discriminate between groups while the most reliable genes for this differentiation were ISG15, MX1 and MX2. MX2 proved to be the best gene for discriminating pregnant buffaloes using the discriminant analysis, while MX1 was the gene that best predicted embryo mortality. Our results showed that among PAG-1, IFNt and ISGs expression as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows, ISGs proved to be the best peripheral biomarkers for predicting pregnancy and embryonic mortality during the peri-implantation period. These insights into the mechanisms behind maternal-fetal interaction and the development of a method for the early detection of embryo distress may enable us to implement effective strategies to support embryo survival.

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

Indoleamine 2, 3-dioxygenase 1 mediated alterations in the functionality of immune cells, deciphers the pregnancy outcomes in crossbred dairy cows

Pregnancy establishment in bovines requires maternal immune cell modulation. Present study investigated possible role of immunosuppressive indolamine-2, 3-dioxygenase 1 (IDO1) enzyme in the alteration of neutrophil (NEUT) and peripheral blood mononuclear cells (PBMCs) functionality of crossbred cows. Blood was collected from non-pregnant (NP) and pregnant (P) cows, followed by isolation of NEUT and PBMCs. Plasma pro-inflammatory (IFNγ and TNFα) and anti-inflammatory cytokines (IL-4 and IL-10) were estimated by ELISA and analysis of IDO1 gene in NEUT and PBMCs by RT-qPCR. Neutrophil functionality was assessed by chemotaxis, measuring activity of myeloperoxidase and β-D glucuronidase enzyme and evaluating nitric oxide production. Changes in PBMCs functionality was determined by transcriptional expression of pro-inflammatory (IFNγ, TNFα) and anti-inflammatory cytokine (IL-4, IL-10, TGFβ1) genes. Significantly elevated (P < 0.05) anti-inflammatory cytokines, increased IDO1 expression, reduced NEUT velocity, MPO activity and NO production observed only in P cows. Significantly higher (P < 0.05) expression of anti-inflammatory cytokines and TNFα genes were observed in PBMCs. Study highlights possible role of IDO1 in modulating the immune cell and cytokine activity during early pregnancy and may be targeted as early pregnancy biomarkers.

Effect of storage time on peripheral blood mononuclear cell isolation from blood collected in vacutainer CPT™ tubes

BackgroundClinical trials of novel therapies for the treatment of ulcerative colitis (UC) may benefit from immune cell profiling, however implementation of this methodology is limited in the multicenter trial setting by necessity of timely (within 6 to 8 h) isolation and processing of peripheral blood mononuclear cells (PBMC) from whole blood samples. Becton Dickinson Vacutainer CPT™ Cell Preparation Tubes (CPT™) limit required processing prior to shipping to a central lab to an initial centrifugation step within 24 h of sample collection. As shipping may delay final processing beyond 24 h, we analyzed cell viability and T cell composition in whole blood stored in CPT™ to determine if their use may accommodate processing delays typical for multicenter clinical trials. MethodsWhole blood samples from 3 patients with UC were collected in CPT™ (15 tubes/patient) and PBMC were processed at various timepoints (24–96 h). Cell viability and T cell composition (26 types) were evaluated by flow cytometry. Variability between technical and biological replicates was evaluated in the context of cell-type abundance, delayed processing time, and data normalization. ResultsTotal cell viability was <50% when processing was delayed to 48 h after collection and was further reduced at later processing timepoints. The effect of delayed processing on cell abundance varied widely across cell types, with CD4+, CD8+, naïve effector CD8+, and Tcm CD4 + T cells displaying the least variability in abundance with delayed processing. Normalization of cell counts to cell types other than total T cells corrected for the effect of delayed processing for several cell types, particularly Th17. ConclusionsBased on these data, processing of PBMC in CPT™ should ideally be performed within 48 h. Delayed processing of PBMC in CPT™ may be considered for cell types that are robust to these conditions. Normalization of cell abundance to different parental cell-types may reduce variability in quantitation and should be used in conjunction with the expected effect size to meet the experimental goals of a multicenter clinical trial.