Novel cell therapy with ex vivo cultured peripheral blood mononuclear cells significantly impacts angiogenesis in the murine ischemic limb model.

Introduction: Autologous mononuclear cells (MNCs) have been used in vascular regenerative therapy since the identification of endothelial progenitor cells (EPCs). However, the efficacy of autologous EPC therapy for diseases such as diabetes and connective tissue disorders is limited due to deficiencies in the number and function of EPCs. To address this, we developed a novel RE-01 cells that enriches pro-angiogenic cells from peripheral blood MNCs (PBMNCs). Methods: PBMNCs were collected from healthy volunteers following ethical guidelines. RE-01 cells were cultured in the presence of specific growth factors for 5 days without media change. Flow cytometry was used to analyze cell surface markers. Tube formation assays, EPC culture assays, and mRNA analysis were performed to evaluate angiogenic potential. The efficacy of RE-01 cells upon transplantation into ischemic hind limbs of mice was evaluated. Results: RE-01 cells exhibited a significant increase in pro-angiogenic cells such as M2 macrophages and angiogenic T cells, in contrast to PBMNCs, while the number of inflammatory cells reduced. In vitro assays demonstrated the enhanced angiogenic abilities of RE-01 cells, supported by increased mRNA expression of angiogenesis-related cytokines. In vivo studies using mouse ischemic hind limb models have shown that blood flow and angiogenesis improved following RE-01 cell transplantation. Transplantations for 3 consecutive days significantly improved the number of pericyte-recruited vessels in the severely ischemic hind limbs of mice. Conclusions: RE-01 cells showed promising results in enhancing angiogenesis and arteriogenesis, possibly owing to the presence of M2 macrophages and angiogenic T cells. These cells also demonstrated anti-fibrotic effects. The efficacy of RE-01 cells has been confirmed in mouse models, suggesting their potential for treating ischemic vascular diseases. Clinical trials are planned to validate the safety and efficacy of RE-01 cell therapy in patients with connective tissue disease and unhealed ulcers. We hope that this new RE-01 cell therapy will prevent many patients from undergoing amputation.

[Establishment of a humanized mouse model of HIV-1 infection and quantification of integrated HIV proviral DNA in vivo].

In recent years, virological, pathological, and immunological studies need to be carried out for the emerging anti-human immunodeficiency virus (HIV) therapies such as gene therapy, broadly neutralizing antibodies, and the derived chimeric antigen receptor (CAR)-T immunotherapy, which necessitates suitable, simple, and inexpensive small-animal models and methods for accurate quantification of the viral genome in the HIV-1 infected. In our research, the HIV-∆ENV-Jurkat-EGFP-mCherry cell line was engineered through the infection with a dual-labelled HIV pseudovirus. A nested quantitative PCR (nested-qPCR) method with the cellular genome as the integrated standard was established for the quantification of HIV proviral copies. We administered intravenous injections of healthy human peripheral blood mononuclear cell (PBMC) into NOD/Prkdcscid/IL2rgnull (NPG) mice. To verify engraftment kinetics, we analyzed the percentages of hCD45+, hCD3+, hCD4+, and hCD8+ cells in the peripheral blood of hu-PBMC-NPG mice. To evaluate HIV-1 infection in hu-PBMC-NPG mice, we inoculated these mice with HIV NL4-3-NanoLuc by intraperitoneal (IP) injection. We then monitored the luciferase expression by the small animal imaging system and measured the viral load in the spleen by qPCR. The infiltration of human PBMCs in mice was detected 3-5 weeks after intravenous injection, and the percentage of hCD45 in humanized mouse PBMCs were more than 25% five weeks after IP inoculation. The expression of the virus-associated luciferase protein was detected by luciferase imaging 27 days post infection. Moreover, the viral total DNA, RNA, and proviral DNA copies reached 18 000 copies/106 cells, 15 000 copies/µg RNA, and 15 000 copies/106 cells, respectively, in the mouse spleen. Taken together, we reported a convenient method for building a simple humanized mouse model of HuPBMC-NPG/severe combined immunodeficiency (SCID) by intravenous injection with hu-PBMCs without advanced surgical skills and irradiation. Furthermore, we established a convenient method for the efficient determination of proviral DNA to assess HIV replication in vivo, viral reservoir sizes, and efficacy of novel anti-HIV therapies including CAR-T immunotherapy and gene therapy.

Technical considerations and strategies for generating and optimizing humanized mouse tumor models in immuno-oncology research.

The field of cancer immunotherapy has experienced significant progress, resulting in the emergence of numerous biological drug candidates requiring in vivo efficacy testing and a better understanding of their mechanism of action (MOA). Humanized immune system (HIS) models are valuable tools in this regard. However, there is a lack of systematic guidance on HIS modeling. To address this issue, the present study aimed to establish and optimize a variety of HIS models for immune-oncology (IO) study, including genetically engineered mouse models and HIS models with human immune components reconstituted in severely immunocompromised mice. The efficacy and utility of these models were tested with several marketed or investigational IO drugs according to their MOA, followed by immunophenotypic analysis and efficacy evaluation. The results of the present study demonstrated that the HIS models responded to various IO drugs as expected and that each model had unique niches, utilities and limitations. Researchers should carefully choose the appropriate models based on the MOA and the targeted immune cell populations of the investigational drug. The present study provides valuable methodologies and actionable technical guidance on designing, generating or utilizing appropriate HIS models to address specific questions in translational IO.

Peripheral Blood Mononuclear Cell Expression of Cation-Chloride Cotransporter (CCC) Genes in Premenstrual Dysphoric Disorder (PMDD) across the Menstrual Cycle-A Preliminary Study.

Premenstrual Dysphoric Disorder (PMDD) is a psychiatric condition characterized by debilitating affective symptomatology in the luteal phase of the menstrual cycle. Based on the previous reports that PMDD may be related to GABAergic cellular dysfunction(s), we assessed whether cation-chloride cotransporter (CCC) gene expression across the menstrual cycle is altered in PMDD. As there are limitations in accessing the human CNS to study CCC-encoding genes, we utilized peripheral blood mononuclear cells (PBMCs) as an alternative model. We first sought to replicate previous reports characterizing CCC gene expression patterns in PBMCs of reproductive age women. We subsequently investigated potential distinct CCC mRNA expression patterns in women with PMDD. We collected blood samples across 8 menstrual cycle visits for PBMC separation/RNA extraction to study mRNA expression of four KCCs (KCC1, KCC2, KCC3, KCC4) and two NKCCs (NKCC1, NKCC2) cotransporters. We mostly replicated the earlier gene expression pattern findings, and found that the expression levels of KCC1 were significantly downregulated during the mid-follicular and periovulatory subphases of the menstrual cycle in women with PMDD. The present study shows that PBMCs is a valid model for studying GABAergic mechanisms underlying PMDD.

Long non-coding RNAs in biomarking COVID-19: a machine learning-based approach.

BACKGROUND: The coronavirus pandemic that started in 2019 has caused the highest mortality and morbidity rates worldwide. Data on the role of long non-coding RNAs (lncRNAs) in coronavirus disease 2019 (COVID-19) is scarce. We aimed to elucidate the relationship of three important lncRNAs in the inflammatory states, H19, taurine upregulated gene 1 (TUG1), and colorectal neoplasia differentially expressed (CRNDE) with key factors in inflammation and fibrosis induction including signal transducer and activator of transcription3 (STAT3), alpha smooth muscle actin (α-SMA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) in COVID-19 patients with moderate to severe symptoms. METHODS: Peripheral blood mononuclear cells from 28 COVID-19 patients and 17 healthy controls were collected. The real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the expression of RNAs and lncRNAs. Western blotting analysis was also performed to determine the expression levels of STAT3 and α-SMA proteins. Machine learning and receiver operating characteristic (ROC) curve analysis were carried out to evaluate the distinguishing ability of lncRNAs. RESULTS: The expression levels of H19, TUG1, and CRNDE were significantly overexpressed in COVID-19 patients compared to healthy controls. Moreover, STAT3 and α-SMA expression levels were remarkedly increased at both transcript and protein levels in patients with COVID-19 compared to healthy subjects and were correlated with Three lncRNAs. Likewise, IL-6 and TNF-α were considerably upregulated in COVID-19 patients. Machine learning and ROC curve analysis showed that CRNDE-H19 panel has the proper ability to distinguish COVID-19 patients from healthy individuals (area under the curve (AUC) = 0.86). CONCLUSION: The overexpression of three lncRNAs in COVID-19 patients observed in this study may align with significant manifestations of COVID-19. Furthermore, their co-expression with STAT3 and α-SMA, two critical factors implicated in inflammation and fibrosis induction, underscores their potential involvement in exacerbating cardiovascular, pulmonary and common symptoms and complications associated with COVID-19. The combination of CRNDE and H19 lncRNAs seems to be an impressive host-based biomarker panel for screening and diagnosis of COVID-19 patients from healthy controls. Research into lncRNAs can provide a robust platform to find new viral infection-related mediators and propose novel therapeutic strategies for viral infections and immune disorders.

DNA methylation in peripheral blood is associated with renal aging and renal function decline: a national community study.

BACKGROUND: Older patients are at risk for acute kidney injury and chronic kidney disease. Age-related increases in DNA methylation at CpG islands have been linked to aging-related diseases like cancer and cardiovascular disease, but the exact causal relationship between methylation in renal aging and other kidney diseases remains unclear. This study aimed to elucidate the methylation status of peripheral blood mononuclear cells (PBMCs) in the Asian population. Using human whole blood DNA methylation analysis from the Taiwan Biobank, we included participants with both whole blood genome-wide methylation data and follow-up data on serum creatinine. We investigated hyper- and hypomethylated genes in comparison of participants with higher and lower estimated glomerular filtration (eGFR) decline rate in overall cohort as well as in comparison of old and young participants in subgroup of participants with higher eGFR decline rate. Common genes and signaling pathways in both comparative analyses were identified. RESULTS: Among 1587 participants in the analysis, 187 participants had higher eGFR decline rate. According to the comparison of methylation in participants with different eGFR declines and at different ages, respectively, we identified common hypermethylated genes, including DNMT3A and GGACT, as well as hypomethylated genes such as ARL6IP5, CYB5D1, BCL6, RPRD2, ZNF451, and MIAT in both participants with higher eGFR decline and those of older age. We observed associations between the methylation status of signaling pathways and aging as well as renal function decline. These pathways notably included autophagy, p38 mitogen-activated protein kinases, and sirtuins, which were associated with autophagy process and cytokine production. CONCLUSIONS: Through methylation analysis of PBMCs, we identified genes and signaling pathways which could play crucial roles in the interplay of renal aging and renal function decline. These findings contribute to the development of novel biomarkers for identifying at-risk groups and even for therapeutic agent discovery.

An Exercise Immune Fitness Test to Unravel Disease Mechanisms-A Proof-of-Concept Heart Failure Study.

Background: Cardiorespiratory fitness positively correlates with longevity and immune health. Regular exercise may provide health benefits by reducing systemic inflammation. In chronic disease conditions, such as chronic heart failure and chronic fatigue syndrome, mechanistic links have been postulated between inflammation, muscle weakness, frailty, catabolic/anabolic imbalance, and aberrant chronic activation of immunity with monocyte upregulation. We hypothesize that (1) temporal changes in transcriptome profiles of peripheral blood mononuclear cells during strenuous acute bouts of exercise using cardiopulmonary exercise testing are present in adult subjects, (2) these temporal dynamic changes are different between healthy persons and heart failure patients and correlate with clinical exercise-parameters and (3) they portend prognostic information. Methods: In total, 16 Heart Failure (HF) patients and 4 healthy volunteers (HV) were included in our proof-of-concept study. All participants underwent upright bicycle cardiopulmonary exercise testing. Blood samples were collected at three time points (TP) (TP1: 30 min before, TP2: peak exercise, TP3: 1 h after peak exercise). We divided 20 participants into 3 clinically relevant groups of cardiorespiratory fitness, defined by peak VO2: HV (n = 4, VO2 ≥ 22 mL/kg/min), mild HF (HF1) (n = 7, 14 < VO2 < 22 mL/kg/min), and severe HF (HF2) (n = 9, VO2 ≤ 14 mL/kg/min). Results: Based on the statistical analysis with 20-100% restriction, FDR correction (p-value 0.05) and 2.0-fold change across the three time points (TP1, TP2, TP3) criteria, we obtained 11 differentially expressed genes (DEG). Out of these 11 genes, the median Gene Expression Profile value decreased from TP1 to TP2 in 10 genes. The only gene that did not follow this pattern was CCDC181. By performing 1-way ANOVA, we identified 8/11 genes in each of the two groups (HV versus HF) while 5 of the genes (TTC34, TMEM119, C19orf33, ID1, TKTL2) overlapped between the two groups. We found 265 genes which are differentially expressed between those who survived and those who died. Conclusions: From our proof-of-concept heart failure study, we conclude that gene expression correlates with VO2 peak in both healthy individuals and HF patients, potentially by regulating various physiological processes involved in oxygen uptake and utilization during exercise. Multi-omics profiling may help identify novel biomarkers for assessing exercise capacity and prognosis in HF patients, as well as potential targets for therapeutic intervention to improve VO2 peak and quality of life. We anticipate that our results will provide a novel metric for classifying immune health.

Low energy availability increases immune cell formation of reactive oxygen species and impairs exercise performance in female endurance athletes.

INTRODUCTION: The effects of low energy availability (LEA) on the immune system are poorly understood. This study examined the effects of 14 days of LEA on immune cell redox balance and inflammation at rest and in response to acute exercise, and exercise performance in female athletes. METHODS: Twelve female endurance athletes (age: 26.8 ± 3.4 yrs, maximum oxygen uptake (V˙O2max): 55.2 ± 5.1 mL × min-1 × kg-1) were included in a randomized, single-blinded crossover study. They were allocated to begin with either 14 days of optimal energy availability diet (OEA, 52 ± 2 kcal × kg fat free mass (FFM)-1 × day-1) or LEA diet (22 ± 2 kcal × kg FFM-1 × day-1), followed by 3 days of refueling (OEA) with maintained training volume. Peripheral blood mononuclear cells (PBMCs) were isolated, and plasma obtained at rest before and after each dietary period. The PBMCs were used for analysis of mitochondrial respiration and H2O2 emission and specific proteins. Exercise performance was assessed on cycle by a 20-min time trial and time to exhaustion at an intensity corresponding to ∼110 % V˙O2max). RESULTS: LEA was associated with a 94 % (P = 0.003) increase in PBMC NADPH oxidase 2 protein content, and a 22 % (P = 0.013) increase in systemic cortisol. LEA also caused an alteration of several inflammatory related proteins (P < 0.05). Acute exercise augmented H2O2 emission in PBMCs (P < 0.001) following both OEA and LEA, but to a greater extent following LEA. LEA also reduced the mobilization of white blood cells with acute exercise. After LEA, performance was reduced in both exercise tests (P < 0.001), and the reduced time trial performance remained after the 3 days of refueling (P < 0.001). CONCLUSION: 14 days of LEA in female athletes increased cortisol levels and had a pronounced effect on the immune system, including increased capacity for ROS production, altered plasma inflammatory proteome and lowered exercise induced mobilization of leukocytes. Furthermore, LEA resulted in a sustained impairment in exercise performance.

Cepharanthine synergistically promotes methylprednisolone pharmacodynamics against human peripheral blood mononuclear cells possibly via regulation of P-glycoprotein/glucocorticoid receptor translocation.

BACKGROUND: Cepharanthin® alone or in combination with glucocorticoid (GC) has been used to treat chronic immune thrombocytopenia (ITP) since the 1990s. Cepharanthine (CEP) is one of the main active components of Cepharanthin®. The purpose of this study was to investigate the effects of CEP on GC pharmacodynamics on immune cells and analyse the possible action mechanism of their interactions. METHODS: Peripheral blood mononuclear cells (PBMCs), T lymphocytic leukemia MOLT-4 cells and daunorubicin resistant MOLT-4 cells (MOLT-4/DNR) were used to evaluate the pharmacodynamics and molecular mechanisms. Drug pharmacodynamics was evaluated by WST-8 assay. P-glycoprotein function was examined by rhodamine 123 assay. CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2/Th17 cytokines were detected by flow cytometry. P-glycoprotein expression and GC receptor translocation were examined by Western blot. RESULTS: CEP synergistically increased methylprednisolone (MP) efficacy with the suppressive effect on the cell viability of PBMCs. 0.3 and 1 µM of CEP significantly inhibited P-glycoprotein efflux function of CD4+ cells, CD8+ cells, and lymphocytes (P<0.05). 0.03~3 µM of CEP also inhibited the P-glycoprotein efflux function in MOLT-4/DNR cells in a concentration-dependent manner (P<0.001). However, 0.03~3 µM of CEP did not influence P-glycoprotein expression. 0.03~0.3 µM of CEP significantly increased the GC receptor distribution from the cytoplasm to the nucleus in a concentration-dependent manner in MOLT-4/DNR cells. The combination did not influence the frequency of CD4+, CD4+CD25+ and CD4+CD25+Foxp3+ T cells or the secretion of Th1/Th2/Th17 cytokines from PBMCs. In contrast, CEP alone at 1 µM decreased the percentage of CD4+ T cell significantly (P<0.01). It also inhibited the secretion of IL-6, IL-10, IL-17, TNF-α, and IFN-γ. CONCLUSIONS: CEP synergistically promoted MP pharmacodynamics to decrease the cell viability of the mitogen-activated PBMCs, possibly via inhibiting P-glycoprotein function and potentiating GC receptor translocation. The present study provides new evidence of the therapeutic effect of Cepharanthin® alone or in combination with GC for the management of chronic ITP.

Impact of a varied set of stimuli on a suite of immunological parameters within peripheral blood mononuclear cells: toward a non-animal approach for assessing immune modulation by materials intended for human use.

Introduction: In toxicology, steps are being taken towards more mechanism-focused and human relevant approaches to risk assessment, requiring new approaches and methods. Additionally, there is increasing emphasis by regulators on risk assessment of immunotoxicity. Methods: Here we present data from a peripheral blood mononuclear cell (PBMC) system whereby a varied set of stimuli, including those against the TCR and Toll-like receptors, enable readouts of cytokine and prostaglandin E2 (PGE2) production with monocyte, T cell and B cell viability, proliferation, and associated activation markers. In addition to results on the impact of the stimuli used, initial profiling data for a case study chemical, curcumin, is presented, illustrating how the system can be used to generate information on the impact of exogenous materials on three major constituent immune cell subsets for use in risk assessment and to direct follow-on studies. Results: The different stimuli drove distinct responses, not only in relation to the "quantity" of the response but also the "quality". Curcumin had a limited impact on the B cell parameters measured, with the stimuli used, and it was noted that in contrast to T cells where there was either no impact or a reduction in viability and proliferation with increasing concentration, for B cells there was a small but significant increase in both measurements at curcumin concentrations below 20 µM. Similarly, whilst expression of activation markers by T cells was reduced by the highest concentration of curcumin, they were increased in B cells. Curcumin only impacted the viability of stimulated monocytes at the highest concentration and had differential impact on different activation markers. Levels of all cytokines and PGE2 were reduced at higher concentrations. Discussion: Although the platform has certain limitations, it nevertheless enables assessment of healthy baseline monocyte, T-, and B-cell responses, and scrutiny of the impact of different stimuli to detect potential immune suppression or enhancement from exogenous materials. In the case of curcumin, a pattern of responses indicative of immune suppressive / anti-inflammatory effects was detected. It is an accessible, highly modifiable system that can be used to screen materials and guide further studies, providing a holistic, integrated picture of effects.

The hiPSC-derived corneal endothelial progenitor-like cell recovers the rabbit model of corneal endothelial dystrophy.

INTRODUCTION: Corneal endothelial dysfunction results in cornea opacity, damaging sightedness, and affecting quality of life. A corneal transplant is the current effective intervention. Due to the scarcity of donated cornea, such an unmet medical need requires a novel therapeutic modality. OBJECTIVES: Customizing patients' corneal endothelial progenitor cells with proliferative activity and lineage restriction properties shall offer sufficient therapeutic cells for corneal endothelial dystrophy. METHODS: The customized induced human corneal endothelial progenitor-like cell (iHCEPLC) was obtained through cell fate conversions starting from PBMC (peripheral blood mononuclear cell), hiPSC (human induced pluripotent stem cell), and hNCC (human neural crest cell), while it finally reached the iHCEPLC state via a series of induction. Several molecular diagnoses were applied to depict its progenitor state, including RNAseq, FlowCytometer, immunostainings, and rtPCR. Significantly, it can be induced to gain differentiation maturity through contact inhibition. In addition, a BAK-mediated rabbit model of corneal endothelial dystrophy was established in the present study to test the therapeutic effectiveness of the iHCEPLC. RESULTS: After inducing cell fate conversion, the specific HCEC markers were detected by rtPCR and immunostaining in iHCEPLC. Further, RNAseq was applied to distinguish its progenitor-like cell fate from primary human corneal endothelial cells (HECE). FlowCytometry profiled the heterogeneity subpopulation, consistently displaying a subtle difference from primary HCEC. A terminal differentiation can be induced in iHCEPLC, addressing its progenitor-like fate. iHCEPLC can restore the BAK-based rabbit model of corneal endothelial dystrophy. Immunohistochemistry verified that such acuity restoration of the BAK-treated cornea is due to the introduced iHCEPLC, and such therapeutic effectiveness is observed in the long term. CONCLUSION: Here, we demonstrated that customized iHCEPLC has long-term therapeutic efficacy. As a progenitor cell, our iHCEPLC has a restricted cell lineage nature and can proliferate in vitro, supporting sufficient therapeutic candidate cells. Due to the immune-privileged nature of the cornea, our iHCEPLC proves the principle of therapeutical feasibility in both autogenic and allogeneic modalities.

SARS-CoV-2 cellular and humoral responses in vaccine-naive individuals during the first two waves of COVID-19 infections in the southern region of The Netherlands: a cross-sectional population-based study.

With the emergence of highly transmissible variants of concern, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still poses a global threat of coronavirus disease 2019 (COVID-19) resurgence. Cellular responses to novel variants are more robustly maintained than humoral responses, and therefore, cellular responses are of interest in assessing immune protection against severe disease in the population. We aimed to assess cellular responses to SARS-CoV-2 at the population level. IFNγ (interferon γ) responses to wild-type SARS-CoV-2 were analyzed using an ELISpot assay in vaccine-naive individuals with different humoral responses: Ig (IgM and/or IgG) seronegative (n = 90) and seropositive (n = 181) with low (<300 U/mL) or high (≥300 U/mL) humoral responses to the spike receptor binding domain (anti-S-RBD). Among the seropositive participants, 71.3% (129/181) were IFNγ ELISpot positive, compared to 15.6% (14/90) among the seronegative participants. Common COVID-19 symptoms such as fever and ageusia were associated with IFNγ ELISpot positivity in seropositive participants, whereas no participant characteristics were associated with IFNγ ELISpot positivity in seronegative participants. Fever and/or dyspnea and anti-S-RBD levels were associated with higher IFNγ responses. Symptoms of more severe disease and higher anti-S-RBD responses were associated with higher IFNγ responses. A significant proportion (15.6%) of seronegative participants had a positive IFNγ ELISpot. Assessment of cellular responses may improve estimates of the immune response to SARS-CoV-2 in the general population. IMPORTANCE: Data on adaptive cellular immunity are of interest to define immune protection against severe acute respiratory syndrome coronavirus 2 in a population, which is important for decision-making on booster-vaccination strategies. This study provides data on associations between participant characteristics and cellular immune responses in vaccine-naive individuals with different humoral responses.

Myeloid-T cell interplay and cell state transitions associated with checkpoint inhibitor response in melanoma.

BACKGROUND: The treatment of melanoma, the deadliest form of skin cancer, has greatly benefited from immunotherapy. However, many patients do not show a durable response, which is only partially explained by known resistance mechanisms. METHODS: We performed single-cell RNA sequencing of tumor immune infiltrates and matched peripheral blood mononuclear cells of 22 checkpoint inhibitor (CPI)-naive stage III-IV metastatic melanoma patients. After sample collection, the same patients received CPI treatment, and their response was assessed. FINDINGS: CPI responders showed high levels of classical monocytes in peripheral blood, which preferentially transitioned toward CXCL9-expressing macrophages in tumors. Trajectories of tumor-infiltrating CD8+ T cells diverged at the level of effector memory/stem-like T cells, with non-responder cells progressing into a state characterized by cellular stress and apoptosis-related gene expression. Consistently, predicted non-responder-enriched myeloid-T/natural killer cell interactions were primarily immunosuppressive, while responder-enriched interactions were supportive of T cell priming and effector function. CONCLUSIONS: Our study illustrates that the tumor immune microenvironment prior to CPI treatment can be indicative of response. In perspective, modulating the myeloid and/or effector cell compartment by altering the described cell interactions and transitions could improve immunotherapy response. FUNDING: This research was funded by Roche Pharma Research and Early Development.

Efficient generation of induced pluripotent stem cell lines from healthy donors' peripheral blood mononuclear cells of different genders.

Peripheral blood mononuclear cell (PBMC) are recognized as a conveniently collected reprogramming resource. Several methods are available in academia to reprogram PBMC into induced pluripotent stem cells (iPSC). In this research, we reprogrammed PBMC of different genders by using non-integrative non-viral liposome electrotransfer containing the reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The three obtained iPSC cell lines were karyotypically normal and showed significant tritiated differentiation potential in vitro and in vivo. Our study provided an efficient procedure for reprogramming PBMC into iPSC and obtained three well-functioning iPSC, that may contribute to advance personalized cell therapy in the future.

Angiogenesis-related immune response may be the prelude to the syndesmophyte formation in Ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic autoimmune arthritis that mainly affects spine joints. To date, the pathogenesis of AS remains unclear, although immune cells and innate immune response cytokines have been suggested to be crucial players. METHODS: By adopting a single-cell RNA sequencing approach in the AS cynomolgus model, we profiled and characterized PBMC proportions along disease progression. RESULTS: Here, our primary focus was on the activation of an immune cascade-initiating lymphocyte subtype known as CD4+CXCR5+ T follicular helper (Tfh) cells. These Tfhs demonstrated a localized residence in AS bone lesion as an ectopic lymphoid structure. Moreover, Tfhs would serve as an upstream initiator for a pro-angiogenic cascade. Then, an expansion in CD14+ monocytes and DC cells subsets resulted in enhanced expression of angiogenesis genes in these AS cynomolgus monkeys. With a confirmed higher abundance of TNF-α accompanying H-type vascular invasion in the osteophytic region, pronounced expansion of Tfhs at such lesion site signaling for monocytes and DCs intrusion is considered as the prelude to the characteristic angiogenic bony outgrowth in AS known as syndesmophytes. CONCLUSIONS: We explored the intimate relationship between local inflammation and bone formation in AS from the perspective of nascent vascularisation. Hence, our study lays the foundation for elucidating a unified AS pathogenesis through the immune-angiogenesis-osteogenesis axis.

Formation of Mycobacterium abscessus colonies in cellular culture in an in vitro infection model.

Mycobacterium abscessus is one of the most important nontuberculous mycobacteria that cause lung diseases. In vitro infection models developed to analyze the immune response are frequently based on the addition of mycobacteria to mononuclear cells or neutrophils from peripheral blood. An important requirement of these assays is that most cells phagocytose mycobacteria, only accomplished by using large multiplicities of infection (1 or more bacteria per cell) which may not adequately reflect the inhalation of a few mycobacteria by the host. We propose modifications that try to mimic some of the conditions in which immune cells deal with mycobacteria. For the preparation of the inoculum mycobacteria are grown in solid media followed by preparation to a single cell suspension. Multiplicities of infection (number of bacteria per cell) are below 0.01. Serum-free cellular media is used to allow the growth of M. abscessus. After several days of incubation Bacterial Colonies in Cellular Culture (BCCC) develop, which are enumerated directly under an inverted microscope. These colonies may represent biofilm formation during chronic infections. •Low multiplicity of infection (below 0.01 bacteria per cell) reflects more realistically conditions encountered by immune cells in the lungs.•The surface of mycobacteria prepared for infection assays that are grown in solid media are less affected than that of mycobacteria grown in liquid media with detergents.•Colony formation in the infected cells may reflect the aggregation and biofilm formation in the lungs during chronic infection.

Uterine infusion strategies for infertile patients with recurrent implantation failure: a systematic review and network meta-analysis.

BACKGROUND: Intra-uterine infusion treatments were reported to be beneficial to embryo implantation and pregnancy outcomes, and considered as potential therapies for infertile patients with recurrent implantation failure (RIF). Nevertheless, their efficiencies were controversial and there lack of consensus on which intrauterine treatment is the most effective. METHODS: All prospective trials (in Chinese or English) were searched in Databases PubMed, Cochrane, Web of Science, and CNKI from July 2013 to July 2023. We included studies that investigated various uterine infusions, including chorionic gonadotropin, granulocyte colony-stimulating factor, monocytes, platelet-rich plasma, etc. during IVF treatment and reported subsequent pregnancy outcomes. RESULTS: We finally included 56 researches, including 40 randomized controlled trials, 14 non-randomized controlled trials, and 3 prospective cohort studies. This study included a total of 11 uterine perfusion methods: Placebo, Human Chorionic Gonadotropin (HCG), Granulocyte Colony-Stimulating Factor (G-CSF), platelet-rich plasma (PRP), Peripheral Blood Mononuclear Cell (PBMC), Growth hormone (GH), dexamethasone (DEX), Embryo culture supernatant (ESC), PRP combined with G-CSF (PRP + G-CSF), RPR combined with subcutaneous injection of G-CSF (RPR + G-CSFsc), G-CSF combined with subcutaneous injection of AXaIU (G-CSF + AXaIUsc). Intrauterine infusion of HCG, PBMC, G-CSF, and PRP significantly improves pregnancy outcomes in patients with repeated implantation failure compared with blank controls or placebo, and PRP improved the clinical pregnancy and live birth most. GH and ESC infusion might improve the pregnancy outcomes, but uterine infusion of DEX was shown with high miscarriage. The combination therapy did not show a significant advantage over the mono-therapy. CONCLUSIONS: Intrauterine infusion of HCG, PBMC, G-CSF, and PRP are promising strategies for improving pregnancy outcomes for infertile patients with recurrent implantation failure. Among these treatments, PRP may be the best. More researches are required to explore the effect of drug combinations and less commonly used drugs as well. TRIAL REGISTRATION: Our study was registered in PROSPERO and the ID was CRD42023467188.

Against all odds: The road to success in the development of human immune reconstitution mice.

The mouse genome has a high degree of homology with the human genome, and its physiological, biochemical, and developmental regulation mechanisms are similar to those of humans; therefore, mice are widely used as experimental animals. However, it is undeniable that interspecies differences between humans and mice can lead to experimental errors. The differences in the immune system have become an important factor limiting current immunological research. The application of immunodeficient mice provides a possible solution to these problems. By transplanting human immune cells or tissues, such as peripheral blood mononuclear cells or hematopoietic stem cells, into immunodeficient mice, a human immune system can be reconstituted in the mouse body, and the engrafted immune cells can elicit human-specific immune responses. Researchers have been actively exploring the development and differentiation conditions of host recipient animals and grafts in order to achieve better immune reconstitution. Through genetic engineering methods, immunodeficient mice can be further modified to provide a favorable developmental and differentiation microenvironment for the grafts. From initially only being able to reconstruct single T lymphocyte lineages, it is now possible to reconstruct lymphoid and myeloid cells, providing important research tools for immunology-related studies. In this review, we compare the differences in immune systems of humans and mice, describe the development history of human immune reconstitution from the perspectives of immunodeficient mice and grafts, and discuss the latest advances in enhancing the efficiency of human immune cell reconstitution, aiming to provide important references for immunological related researches.

Single-cell RNA sequencing reveals cell type-specific immune regulation associated with human neuromyelitis optica spectrum disorder.

Introduction: One rare type of autoimmune disease is called neuromyelitis optica spectrum disorder (NMOSD) and the peripheral immune characteristics of NMOSD remain unclear. Methods: Here, single-cell RNA sequencing (scRNA-seq) is used to characterize peripheral blood mononuclear cells from individuals with NMOSD. Results: The differentiation and activation of lymphocytes, expansion of myeloid cells, and an excessive inflammatory response in innate immunity are observed. Flow cytometry analyses confirm a significant increase in the percentage of plasma cells among B cells in NMOSD. NMOSD patients exhibit an elevated percentage of CD8+ T cells within the T cell population. Oligoclonal expansions of B cell receptors are observed after therapy. Additionally, individuals with NMOSD exhibit elevated expression of CXCL8, IL7, IL18, TNFSF13, IFNG, and NLRP3. Discussion: Peripheral immune response high-dimensional single-cell profiling identifies immune cell subsets specific to a certain disease and identifies possible new targets for NMOSD.

Successful wound healing by autologous peripheral blood mononuclear cell therapy in a diabetic patient on hemodialysis with no-option critical limb ischemia: a case report.

Peripheral artery disease is a common condition in patients on chronic dialysis treatment, end-stage kidney failure itself being a risk factor. The most severe stage of peripheral artery disease, critical limb ischemia, causes marked chronic pain and is associated with risk of limb loss. Despite improvements in revascularization procedures, the results of limb salvage procedures among dialysis patients remains poor, and lower extremity amputation is associated with high mortality and grim socio-economic implications. We report on a limb salvage approach that was successfully employed in a 74-year-old woman on hemodialysis suffering from no-option critical limb ischemia complicated by diabetic foot infection, i.e. otherwise a candidate for major amputation. The approach consists in implanting in the wound bed of the affected limb a concentrate of autologous peripheral blood mononuclear cells collected from the peripheral blood of the patient using a selective filtration separation system. The procedure, performed by a vascular surgeon in an outpatient setting and sterile conditions, was repeated three times at intervals of 15 days, and was well tolerated; no adverse safety signals were observed. Complete wound healing was obtained, with successful limb rescue.