Mononuclear Cells Research Articles

α-Synuclein disrupts microglial autophagy through STAT1-dependent suppression of Ulk1 transcription

BackgroundAutophagy dysfunction in glial cells is implicated in the pathogenesis of Parkinson’s disease (PD). The previous study reported that α-synuclein (α-Syn) disrupted autophagy in cultured microglia. However, the mechanism of microglial autophagy dysregulation is poorly understood.MethodsTwo α-Syn-based PD models were generated via AAV-mediated α-Syn delivery into the mouse substantia nigra and striatal α-Syn preformed fibril (PFF) injection. The levels of microglial UNC-51-like kinase 1 (Ulk1) and other autophagy-related genes in vitro and in PD mice, as well as in the peripheral blood mononuclear cells of PD patients and healthy controls, were determined via quantitative PCR, western blotting and immunostaining. The regulatory effect of signal transducer and activator of transcription 1 (STAT1) on Ulk1 transcription was determined via a luciferase reporter assay and other biochemical studies and was verified through Stat1 knockdown or overexpression. The effect of α-Syn on glial STAT1 activation was assessed by immunohistochemistry and western blotting. Changes in microglial status, proinflammatory molecule expression and dopaminergic neuron loss in the nigrostriatum of PD and control mice following microglial Stat1 conditional knockout (cKO) or treatment with the ULK1 activator BL-918 were evaluated by immunostaining and western blotting. Motor behaviors were determined via open field tests, rotarod tests and balance beam crossing.ResultsThe transcription of microglial ULK1, a kinase that controls autophagy initiation, decreased in both in vitro and in vivo PD mouse models. STAT1 plays a critical role in suppressing Ulk1 transcription. Specifically, Stat1 overexpression downregulated Ulk1 transcription, while Stat1 knockdown increased ULK1 expression, along with an increase in LC3II and a decrease in the SQSTM1/p62 protein. α-Syn PFF caused toll-like receptor 4-dependent activation of STAT1 in microglia. Ablation of Stat1 alleviated the decrease in microglial ULK1 expression and disruption of autophagy caused by α-Syn PFF. Importantly, the ULK1 activator BL-918 and microglial Stat1 cKO attenuated neuroinflammation, dopaminergic neuronal damage and motor defects in PD models.ConclusionsThese findings reveal a novel mechanism by which α-Syn impairs microglial autophagy and indicate that targeting STAT1 or ULK1 may be a therapeutic strategy for PD.

The Effect of Copper Nanoparticles on Liver Metabolism Depends on the Type of Dietary Fiber.

A diet enriched with copper nanoparticles (CuNPs) exhibits a wide range of effects on liver metabolism, both positive and negative. Dietary fibers are the key components that may affect the absorption of minerals, including copper, and change their impact on organisms. Therefore, this study investigated whether and how supplementation with different sources of dietary fiber (cellulose, pectin, inulin, and psyllium) affects the function of CuNPs in the liver of male Wistar rats. The results showed that CuNPs at different doses had varying effects on lipid metabolism and inflammation in the liver. Specifically, higher doses of CuNPs were associated with increased lipid accumulation and the activation of pro-inflammatory mechanisms. However, combining CuNPs with dietary fibers, such as psyllium and inulin, was beneficial in mitigating the effects of the examined nanoparticles, leading to reduced fat, cholesterol, and triglycerides in the liver. Combining psyllium with CuNPs showed the most substantial effect on liver metabolism and inflammation parameters. Furthermore, hepatic histology analyses showed that adding psyllium to the diet with CuNPs reduces changes associated with fat accumulation and mononuclear cell infiltration. The observed beneficial changes in the liver may have been related to a reduction in the gene expression level of sterol regulatory element-binding protein 1 and peroxisome proliferator-activated receptor gamma and cyclooxygenase-2. In conclusion, enriching the diet with dietary fibers such as psyllium can regulate the action of CuNPs, thereby improving lipid metabolism and reducing inflammation in the liver.

Diagnosis of canine B-cell chronic lymphoid leukemia with a CD21 negative phenotype using the LT21 clone CD21 antibody in flow cytometry: a case report

BackgroundChronic lymphoid leukemia (CLL) is a hematological disorder characterized by the clonal expansion of small mature lymphocytes that accumulate in the blood and bone marrow. CLL can arise from B-, T-, or natural killer cell clones. The cytological evaluation of blood smears is often the simplest and least invasive method for diagnosing lymphoid leukemia. Immunophenotyping is used to further subclassify the type of lymphoid leukemia.Case presentationA 15-year-old, 4.4-kg spayed female Shih Tzu was presented to the veterinary medical teaching hospital of Kangwon National University. Despite having a normal appetite and activity level, cervical and inguinal lymph node enlargement was noted on physical examination. Complete blood count revealed severe leukocytosis, severe lymphocytosis, and monocytosis. Splenomegaly, hepatomegaly, and lymph node enlargement were detected on radiographic and ultrasonographic examination. Immunophenotyping was performed using peripheral blood mononuclear cells (PBMCs). The majority of lymphocytes exhibited the following profiles: CD3−CD79a− (97.5%), CD4−CD8− (98.6%), CD21−CD79a− (98.4%), CD34− (0.1%), CD45+ (99.6%), major histocompatibility complex class II+ (99.5%), and CD14− (0.5%). Based on the immunophenotyping results, possible differentials considered included the following: the majority of lymphocytes may be natural killer (NK) cell clones, plasma cell clones, or show aberrant expression or loss of CD21 marker due to the neoplastic nature of the cells. Further flow cytometry was performed using antibodies against CD3, CD5, CD94, and granzyme B. The combined results indicated that the predominant lymphocyte subset in the PBMCs was CD3−CD5−CD21−CD94−granzyme B−. To confirm monoclonality and exclude the aberrant loss of CD markers, a polymerase chain reaction for antigen receptor rearrangement (PARR) assay was conducted. The PARR assay, using DNA from blood and lymph node samples, showed B-cell monoclonality. Immunocytochemistry using PBMCs showed that the plasma cell marker Multiple Myeloma Oncogene 1 (MUM1) was not expressed. Therefore, the diagnosis was confirmed to be B-cell CLL.ConclusionImmunophenotyping can help subclassify the type of lymphoid leukemia; however, as tumor cells can show aberrant expression or loss of the CD21 marker, combining immunophenotyping with the PARR assay could yield a more accurate diagnosis.

Tumor organoids improve mutation detection of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) presents challenges in detecting somatic mutations due to its complex cellular composition. This study investigated the utility of patient-derived organoids (PDOs) to overcome these obstacles and enhance somatic mutation identification. Surgically resected PDAC tumors and their paired PDOs from 21 patients were examined. Whole-exome sequencing (WES) of tumor tissue, organoids, and peripheral blood mononuclear cells was performed to identify somatic mutations. Our findings demonstrate that PDOs retained about 80% of the somatic mutations from the original tumors, showing high concordance in mutation types. PDOs exhibited increased tumor purity and uncovered key driver mutations, aiding in identifying clinically relevant genomic alterations. Moreover, eight cycles of FOLFIRINOX treatment did not significantly alter the mutational landscape at the DNA level, indicating the stability of the mutational profile after therapeutic pressure in patients. In conclusion, PDOs are potentially important tools for exploring the somatic mutational landscape of PDAC. While they can reveal mutations that may be challenging to detect through traditional biopsy sequencing due to the inherently low tumor purity of PDAC, it is important to note that PDOs may not always fully recapitulate all mutations found in primary tumors. Despite this limitation, PDOs can still offer critical insights into the genomic complexities of PDAC, which is crucial for the development of personalized vaccines and therapies for this disease.

ALDH2 mediates the effects of sodium-glucose cotransporter 2 inhibitors (SGLT2i) on improving cardiac remodeling

BackgroundSodium-glucose cotransporter-2 inhibitors (SGLT2i) are now recommended for patients with heart failure, but the mechanisms that underlie the protective role of SGLT2i in cardiac remodeling remain unclear. Aldehyde dehydrogenase 2 (ALDH2) effectively prevents cardiac remodeling. Here, the key role of ALDH2 in the efficacy of SGLT2i on cardiac remodeling was studied.MethodsAnalysis of multiple transcriptomic datasets and two-sample Mendelian randomization were performed to find out the differentially expressed genes between pathological cardiac hypertrophy models (patients) and controls. A pathological cardiac hypertrophy mouse model was established via transverse aortic constriction (TAC) or isoproterenol (ISO). Cardiomyocyte-specific ALDH2 knockout mice (ALDH2CMKO) and littermate control mice (ALDH2flox/flox) were generated to determine the critical role of ALDH2 in the preventive effects of dapagliflozin (DAPA) on cardiac remodeling. RNA sequencing, gene knockdown or overexpression, bisulfite sequencing PCR, and luciferase reporter assays were performed to explore the underlying molecular mechanisms involved.ResultsOnly ALDH2 was differentially expressed when the differentially expressed genes obtained via Mendelian analysis and the differentially expressed genes obtained from the multiple transcriptome datasets were combined. Mendelian analysis revealed that ALDH2 was negatively related to the severity of myocardial hypertrophy in patients. DAPA alleviated cardiac remodeling in mouse hearts subjected to TAC or ISO. ALDH2 expression was reduced, whereas ALDH2 expression was restored by DAPA in hypertrophic hearts. Cardiomyocyte specific ALDH2 knockout abolished the protective role of DAPA in preventing cardiac remodeling. ALDH2 expression and activity were increased in DAPA-treated neonatal rat primary cardiomyocytes (NRCMs), H9C2 cells and AC16 cells. Moreover, DAPA upregulated ALDH2 in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes. Sodium/proton exchanger 1 (NHE1) inhibition contributed to the regulation of ALDH2 by DAPA. DAPA suppressed the production of reactive oxygen species (ROS), downregulated DNA methyltransferase 1 (DNMT1) and subsequently reduced the ALDH2 promoter methylation level. Further studies revealed that DAPA enhanced the binding of nuclear transcription factor Y, subunit A (NFYA) to the promoter region of ALDH2, which was due to the decreased promoter methylation level of ALDH2.ConclusionsThe upregulation of ALDH2 plays a critical role in the protection of DAPA against cardiac remodeling. DAPA enhances the binding of NFYA to the ALDH2 promoter by reducing the ALDH2 promoter methylation level through NHE1/ROS/DNMT1 pathway.Graphical abstract

Proteogenomic characterization of highly enriched viable leukemic blasts in acute myeloid leukemia: A SWOG report

AbstractIntroductionAcute myeloid leukemia (AML) remains one of the deadliest hematopoietic malignancies. A better understanding of the molecular biology governing AML may lead to improved risk stratification and facilitate the development of novel therapies. Proteins are responsible for much of the biology of cells. Several studies have examined the global proteome in bulk mononuclear cells (MNCs) from AML specimens, which are comprised a heterogenous population of cells at various stages of differentiation.MethodsGiven the potential impact of the nonleukemic cells on protein expression profiles, we applied an integrative proteogenomic approach utilizing next‐generation sequencing and mass spectrometry‐based proteomics to identify novel protein biomarkers in unsorted MNCs and viable leukemic blasts (VLBs) isolated from blood and bone marrow specimens obtained at the time of AML diagnosis.ResultsWe identified significant differences in protein expression between VLBs and MNCs. Subsequent studies (N = 27) focused on proteomic profiling of VLBs that identified novel candidate biomarkers associated with mutational genotypes and clinical outcome, some of which were recapitulated in an independent cohort of patients. Using mass spectrometry, we also detected mutated protein products, some of which were predicted via in silico analyses to be potential neoantigens amenable to adoptive immunotherapy. As previously described, analyses comparing transcript and protein expression showed an overall modest correlation between mRNA and protein dataset, but enriching for genes associated with mutations significantly improved the protein–RNA correlation.ConclusionTogether, the results provide insight into the biology of VLBs and demonstrate the gains derived from examining the proteome in addition to genome and transcriptome.

Immune alterations in children with autism spectrum disorders

Autism spectrum disorders (ASD) are a heterogeneous group of mental and nervous system disorders. Patients with ASD are characterized by communication and cognitive impairments and obsessive behavior. The pathogenesis and etiology of ASD are still unclear. According to the literature, patients suffering from ASD have not only mental, but also somatic disorders, including changes in the immune system. The aim of this work was to study the concentration of cytokines in the blood plasma of children with ASD and the level of expression of proinflammatory genes in peripheral blood mononuclear cells. The clinical group included 71 children aged 4-12 years with a diagnosis of ASD (F84.02). Patients scored more than 36 on the Childhood Autism Rating Scale (CARS). The control sample included 27 apparently healthy children of the same age. The following methods were used in this study: isolation of mononuclear cells from heparinized peripheral blood, Ficoll-Verografin density gradient centrifugation, evaluation of cytokine blocks using commercially available enzyme immunoassay kits, isolation of random total RNA, reverse transcription using hexaprimers, and real-time polymerase chain reaction using intercalating dye SYBR Green. The concentration of proinflammatory cytokines IL-1β, IL-8, and IL-17A in the peripheral blood plasma of children with ASD was statistically significantly increased compared to the control sample. Moreover, the concentration of the anti-inflammatory cytokine IL-10 in patients with ASD was 3.6 times lower compared to the control sample (p 0.001). The level of expression of the NF-κB1, IL1β, IL8 and TNFα genes at the RNA level in peripheral blood mononuclear cells was increased by 2.8, 2.5, 4.8 and 4.2 times in patients with ASD compared to the control sample (all p 0.01). The results obtained indicate an increase in the concentration of proinflammatory cytokines (IL-1β, IL-8, IL-17A) in the blood plasma and a decrease in the concentration of anti-inflammatory cytokines (IL-10) in patients with ASD compared to the control sequence.

Interactions between the human milk oligosaccharide 2'-fucosyllactose and Bifidobacterium longum subspecies infantis in influencing systemic immune development and function in piglets.

The oligosaccharide 2'-fucosyllactose (2'-FL) is a predominant component of human milk, serving as a prebiotic for gut microbiota and influencing immune development in infants. Bifidobacterium longum subspecies infantis (B. infantis) is a commensal bacterium found in breastfed infants. Both 2'-FL and a specific strain of B. infantis, Bi-26™, are commercially available. This study investigates the potential synbiotic relationship between 2'-FL and Bi-26™ on immune development. Two-day-old piglets (n = 53) were randomized in a 2 × 2 design, receiving either a commercial milk replacer ad libitum without (CON) or with 1.0 g/L 2'-FL (FL). Piglets in each diet were further randomized to receive either glycerol stock alone or Bi-26™ (109 CFU) (BI and FLBI) orally once daily. On postnatal day (PND) 34/35, animals were euthanized, and blood was collected for serum cytokine analysis. Additionally, peripheral blood mononuclear cells (PBMCs) were isolated for ex vivo stimulation and flow cytometry analysis. Serum and ex vivo cytokines were analyzed using a multivariate model. All other outcomes were analyzed using a two-way ANOVA, considering prebiotic and probiotic fixed effects. The significance level was set at a p value <0.05, with trends reported for 0.05 < p < 0.1. Immune cell populations in PBMCs were unaffected by the experimental treatment. However, serum interleukin (IL)-1RA, IL-1β, IL-12, and IL-18 were all higher (p < 0.05) in the FL group than in the CON group. In isolated PBMCs, lipopolysaccharide (LPS) stimulation resulted in higher IL-1RA and a trend for higher IFN-γ secretion in the FL group vs. the CON group. 2'-FL stimulates a balanced cytokine profile in healthy piglets without changing immune cell populations. When immune cells are stimulated ex vivo with LPS, 2'-FL primes T-cells for a proinflammatory response, which is moderated by co-administration of Bi-26™.

Effect of RTS,S/AS01E vaccine booster dose on cellular immune responses in African infants and children

RTS,S/AS01E, the first approved malaria vaccine, demonstrated moderate efficacy during the phase 3 pediatric trial. We previously investigated cell-mediated immune (CMI) responses following the primary 3-dose immunization and now report responses to the booster dose given 18 months later. Thirty CMI markers were measured by Luminex in supernatants of peripheral blood mononuclear cells from 709 children and infants after RTS,S/AS01E antigen stimulation, and their associations with malaria risk and antibodies one month post-booster and one year later were assessed. IL-2, IFN-γ, IL-17, IL-5, and IL-13 were associated with RTS,S/AS01E booster vaccination, and IL-2 responses to the circumsporozoite protein (CSP) remained higher after one year. IL-2 was associated with reduced malaria risk in one site, and IL-10 was associated with increased risk in infants. Anti-CSP IgG and IL-2 were moderately correlated one year after booster. This study highlights the moderate cell-mediated immunogenicity of the RTS,S/AS01E booster dose that aligns with partial recovery of RTS,S/AS01E vaccine efficacy.

Glycogen Storage Disease Type I and Bone: Clinical and Cellular Characterization.

Glycogen storage disease (GSD) is the most prevalent inherited disorder of glycogen metabolism for which no causal treatment is available. In recent years, thanks to the improved clinical management, the life expectancy of these patients extended, disclosing previously unidentified adverse conditions in other organs. In this study, we evaluated the clinical bone complications and the cellular responses in 20 patients (aged 14.1 ± 3.4years) affected by GSD type I. Fragility fractures were reported in 35% of the patients, which were older than unfractured patients. They involved appendicular skeletal segments, while no vertebral deformity was detected. 60% of the patients had a bone mineral density (BMD) "below the expected range for age", and lumbar spine (LS) BMD Z-scores positively correlated with muscle strength. Circulating mineral and bone markers showed reduction in the older subjects, with no increase in the pubertal age. Significant correlations could not be detected between circulating markers and LS BMD Z-scores, except for sclerostin levels, which also correlated with muscle strength. The osteoclasts differentiated from patients' peripheral blood mononuclear cells did not show cell-autonomous alterations. However, circulating osteoclast precursors from healthy individuals cultured in the presence of patients' sera exhibited increased osteoclastogenesis compared to control sera suggesting that GSD type I serum factors could affect osteoclast function in a non-autonomous manner. In contrast, circulating osteoprogenitors were unremarkable.

Single-cell RNA sequencing uncovers molecular mechanisms of intravenous immunoglobulin plus methylprednisolone in Kawasaki disease: attenuated monocyte-driven inflammation and improved NK cell cytotoxicity.

Intravenous immunoglobulin (IVIG) plus methylprednisolone as initial intensive therapy or additional therapy in Kawasaki disease (KD) has been used in clinical practice. However, its molecular and cellular mechanism is unclear. We performed single-cell analysis on 14 peripheral blood mononuclear cell (PBMC) samples obtained from 7 KD patients who received either IVIG monotherapy or IVIG plus methylprednisolone therapy. This encompassed 4 samples from KD patients collected before and after IVIG treatment, as well as 3 samples from KD patients before and after IVIG plus methylprednisolone therapy. Both IVIG monotherapy and IVIG plus methylprednisolone therapy can increase lymphocyte counts (e.g. CD4+T, CD8+T, and gdT cells) to address lymphopenia. They can also decrease monocyte counts and repress the expression of S100A12, NLRP3, and genes associated with immune-cell migration in monocytes. IVIG combined with methylprednisolone downregulates more monocyte-driven inflammatory pathways than IVIG alone. Additionally, this combination uniquely enhances NK cell cytotoxicity by modulating receptor homeostasis, while significantly upregulating interferon-related genes in CD4+ T cells, CD8+ T cells, and B cells, particularly type I interferons. The combination of IVIG with methylprednisolone attenuated monocyte-driven inflammation and improved NK cell cytotoxicity which might provide clues for pediatricians to consider treatment options for children with KD. Whether the monocyte-driven hyperinflammatory state and NK cell function can be indicators for the clinical choice of IVIG with methylprednisolone therapy in KD needs further investigation.

Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners.

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.

Consumption of a Probiotic Blend with Vitamin D Improves Immunity, Redox, and Inflammatory State, Decreasing the Rate of Aging—A Pilot Study

There is evidence of the effect of probiotic intake on the immune system. However, the effect probiotics may have on the rate of aging is unknown. The aim of this study is to determine the effect of a probiotic blend on immunity, redox state, inflammation, and the rate of aging or biological age. A group of 10 men and 14 women took, daily for 2 months, a sachet with three probiotics (Bifidobacterium animalis subsp. lactis BSO1, Lactobacillus reuteri LRE02, Lactobacillus plantarum LP14) and vitamin D. Before starting the treatment and after 2 months, peripheral blood was collected. Immune functions were assessed in isolated immune cells, and cytokine concentrations were also measured both in mononuclear cell cultures and plasma. Redox state parameters were also analyzed in whole blood cells. Finally, the Immunity Clock was applied to determine the biological age. Results show that the intake of this probiotic blend in general, in both men and women, improves immunity and decreases the oxidative and inflammatory state. In addition, it rejuvenates the biological age by 10 years on average. It can be concluded that this probiotic blend could be proposed as a good strategy to slow down the aging process, and to achieve healthy aging.

Dennd2c Negatively Controls Multinucleation and Differentiation in Osteoclasts by Regulating Actin Polymerization and Protrusion Formation.

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages.

IL-27 attenuated macrophage injury and inflammation induced by Mycobacterium tuberculosis by activating autophagy.

Interleukin-27 (IL-27) is a cytokine that is reported to be highly expressed in the peripheral blood of patients with pulmonary tuberculosis (PTB). IL-27-mediated signaling pathways, which exhibit anti- Mycobacterium tuberculosis (Mtb) properties, have also been demonstrated in macrophages infected with Mtb. However, the exact mechanism remains unclear. This study aimed to clarify the potential molecular mechanisms through which IL-27 enhances macrophage resistance to Mtb infection.Both normal and PTB patients provided bronchoalveolar lavage fluid (BALF). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals and stimulated with 50ng/mL macrophage-colony stimulating factor (M-CSF) to obtain monocyte-derived macrophages (MDMs). Using 100ng/mL phorbol 12-myristate 13-acetate (PMA), THP-1 cells were induced to differentiate into THP-1-derived macrophage-like cells (TDMs). Both MDMs and TDMs were subsequently infected with the Mtb strain H37Rv and treated with 50ng/mL IL-27 prior to infection. The damage and inflammation of macrophages were examined using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and Western blotting.Patients with PTB had elevated levels of IL-27 in their BALF. Preconditioning with IL-27 was shown to reduce H37Rv-induced MDMs and TDMs apoptosis while also decreasing the levels of Cleaved Caspase-3, Bax and the proinflammatory cytokines TNF-α, IL-1β, and IL-6, promoting the expression of Bcl-2 and the anti-inflammatory factors IL-10 and IL-4. Silencing of the IL-27 receptor IL-27Ra increased macrophage damage and inflammation triggered by H37Rv. Mechanistically, IL-27 activates autophagy by inhibiting TLR4/NF-κB signaling and activating the PI3K/AKT signaling pathway, thereby inhibiting H37Rv-induced macrophage apoptosis and the inflammatory response.Our study suggests that IL-27 alleviates H37Rv-induced macrophage injury and the inflammatory response by activating autophagy and that IL-27 may be a new target for the treatment of PTB.

Immune Effect of Co-Culture of Dendritic Cells and Cytokine-Induced Killer Cells on Prostate Cancer Cells.

It was to explore the immune outcome of co-culture of dendritic cells (DC) and cytokine-induced killer cells (CIK) on prostate cancer (PCa) cells. Peripheral blood mononuclear cells (PBMCs) were extracted from healthy blood donors. DC and CIK cells were induced and co-cultured. The proliferation and phenotypic changes of DC, CIK, and DC-CIK cells/groups were observed. Model rats were constructed by injecting PC3 PCa cells into the abdominal cavity. The successful 50 cases were divided into a negative control group, a chemotherapy group, a DC group, a CIK group, and a DC-CIK treatment group (each consisting of 10 rats) to observe tumor progression. The secreted concentrations of interleukin-12 (IL-12) ((103.67 ± 2.77) pg/mL) and interferon-γ (IFN-γ) ((730.09 ± 23.52) pg/mL) were higher in DC-CIK group as against DC and CIK groups; the proliferation of CIK was higher in DC-CIK group as against CIK within 12 to 20 days of culture. The positive rate (PR) of CD3+/ CD56+ and CD8+ was higher and that of CD45RA+ was lower in DC-CIK group as against CIK.The killing rate of the DC-CIK group was higher than that of the DC and CIK groups at a target effect ratio of 10:1/20:1/50:1 (P < 0.05). After the treatment, the body weight of rats in the chemotherapy group, DC group, and CIK group was significantly reduced (P < 0.05), while no significant changes were observed in the negative control group and DC-CIK group (P > 0.05). After 25 days of treatment, the tumor size in the DC-CIK group was significantly smaller compared to the negative control group, chemotherapy group, DC group, and CIK group; the necrotic area of the tumor tissue in the DC-CIK group was also significantly larger than that in the negative control group, chemotherapy group, DC group, and CIK group (P < 0.05). Co-culture of DC and CIK is excellent in enhancing the proliferation of CIK cells, increasing the secretion of IL-12 and IFN-γ, and enhancing the activity of immune cells and anti-tumor ability, showing its potential in anti-PCa tumor immunotherapy.

The Effect of Boric Acid and Calcium Fructoborate on T Helper Cell Differentiation by Influencing Foxp3 and Ror-γt in Rheumatoid Arthritis and Systemic Lupus Erythematosus.

Many animal and human studies indicate that boric acid and calcium fructoborate have effects on helper T cells in immunity. The aim of our study is to evaluate the effects of boric acid and calcium fructoborate on Treg (CD4+Foxp3+) and Th17 (CD4+Ror-γt+) cell populations and related cytokine levels in mononuclear cells isolated from peripheral blood samples of rheumatoid arthritis and systemic lupus erythematosus patients. Newly diagnosed rheumatoid arthritis (n = 10) patients, systemic lupus erythematosus (n = 5) patients, and healthy individuals (n = 9) were included in this study. Consent forms were obtained from all individuals participating the study, blood samples were taken, and peripheral blood mononuclear cells were isolated. Isolated cells were exposed to low-dose and high-dose boric acid and calcium fructoborate in cell culture. Treg and Th17 cell populations were analyzed by flow cytometry after 48h of exposure. IL-2, IL-6, IL-17, IL-23, TNF-α, and TGF-β levels in the culture medium were tested by ELISA method. At the end of the study, in healthy controls, high-dose BA improved the Treg/Th17 population but could not display similar effects on RA and SLE group. However, both boric acid and calcium fructoborate at different doses showed an increasing effect on Ror-γt in RA and SLE group. Different doses of BA and CaF treatment found to have a variable effect on cytokine. Both BA and CaF in low doses decreased TNF-α levels in RA group which shows that these boron compounds could contribute positively to the treatment of autoimmune diseases.

Expression of PGC-1α, PPAR-α and UCP1 genes, metabolic and anthropometric factors in response to sodium butyrate supplementation in patients with obesity: a triple-blind, randomized placebo-controlled clinical trial.

There is increasing evidence that gut metabolites have a role in the etiology of obesity. This study aimed to investigate the effects of sodium butyrate (NaB) supplementation on the expression of peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1α (PGC-1α), PPAR-α, and uncoupling protein-1 (UCP-1) genes, as well as on the metabolic parameters and anthropometric indices in persons with obesity. In this triple-blind placebo-controlled randomized clinical trial, 50 individuals with obesity were randomly assigned to NaB (600 mg/day) + hypo-caloric diet or placebo group + hypo-caloric diet for 8 weeks. The study measured the participants' anthropometric characteristics, food consumption, and feelings of hunger in addition to the serum levels of metabolic indices and the mRNA expression of the PGC-1α, PPAR-α, and UCP-1 genes in peripheral blood mononuclear cells (PBMCs). PGC-1α and UCP-1 genes expression significantly increased in NaB group compared to the placebo at the endpoint. A significant decrease in weight, BMI, and waist circumference (WC) was observed in NaB group. Among the metabolic factors, NaB significantly decreased fasting blood sugar (FBS) (P = 0.04), low-density lipoprotein cholesterol (LDL-C) (P = 0.038) and increased high-density lipoprotein cholesterol (HDL-C) (P = 0.016). NaB could not significantly change serum GLP-1 level. This study unveiled NaB supplementation alone cannot have significant beneficial effects on anthropometric, and biochemical factors. NaB could affect anthropometric and metabolic risk variables associated with obesity only when prescribed, along with calorie restriction. This study was registered in the Iranian Registry of Clinical Trials ( https://en.irct.ir/trial/53968 ) on 31 January 2021 (registry number IRCT20190303042905N2).

A New IL-6-Inducing Mechanism in Cancer with New Therapeutic Possibilities.

Background: Interleukin-6 is dysregulated in multiple pathological conditions, e.g., cancer and inflammatory diseases. Aim: To investigate new mechanisms for the regulation of pathological IL-6 production. Methods: PBMCs (peripheral blood mononuclear cells) stimulated by cancer serum factors or specific peptides produce interleukin-6 (IL-6). Immunoregulatory albumin neo-structures and peptides were identified with 2D gel electrophoresis and MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analyses. Il-6 and albumin neo-structures were determined by ELISA (enzyme-linked immunosorbent assay). Results: Conformational changes in normal serum albumin by proteolytic degradation generates an IL-6-inducing neo-structure, IL-6-inducing factor (IL-6IF). This neo-structure is immunogenic which results in the production of autoantibodies. IL-6 production induced by IL-6IF and cancer patient sera is inhibited by specific antibodies. The serum concentration of IL-6IF is significantly higher in advanced cancer stages, and its presence is significantly correlated with the survival of the patients. Conclusions: A new mechanism for the induction IL-6 synthesis is presented. Based on this mechanism, the pathological IL-6 production related to enhanced proteolytic activity can be diagnosed and selectively inhibited by specific antibodies. Such antibodies were identified and purified. Thus, the neo-structure, inducing pathological IL-6 production, associated with a reduced survival of cancer patients, can be selectively removed by the therapeutic administration of antibodies leaving the function of IL-6 needed for the normal activity of the immune system intact.

Chronic heat stress upregulates pyruvate metabolic process and gluconeogenesis but downregulates immune responses in Sahiwal cattle.

Climate change and growing population and their strain on animal production are the impending challenges that the developing countries, like India, need to tackle in the coming days. This study aimed to detect and analyze the uncharacterized variation in the gene expression patterns with the change of condition, from thermoneutral to chronic hot-humid, in the Sahiwal cattle, one of the best breeds of milk-producing cattle in India, known for being heat-tolerant. Using RNA-Seq analysis on peripheral blood mononuclear cells (PBMCs), 4021 differentially expressed mRNAs (2772 upregulated, 1249 downregulated) and 1303 differentially expressed long non-coding RNAs (769 upregulated, 534 downregulated) were identified, with the thresholds of false discovery rate < 0.05 and|log2(fold change)| > 2. Significantly (p-adjusted < 0.05) overrepresented Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathways were analyzed, revealing upregulation of processes like pyruvate metabolic process, gluconeogenesis, ion transmembrane transport, neuropeptide signaling pathway, and animal organ development, with genes like SHH, GRK1, CHRM3, CAMK2A, and HSPB7 were upregulated, while translation and immune responses, with genes like RPS3, EEF1A1, TNF, BoLA-DRB3, and UBB were downregulated. Analysis of cis-mRNAs of DE-lncRNAs showed presence of both up- and down-regulated cis-mRNAs for both up- and down-regulated lncRNAs indicating existence of positive and negative regulation of mRNA expression by lncRNAs. Managemental nudges that decrease metabolic heat generation, like betaine and chromium supplementation, and increase heat dissipation, like microenvironment cooling, should be utilized. This study highlights the role of pyruvate metabolism and gluconeogenesis in coping up with heat stress and offers an improved understanding of the heat stress response of Sahiwal cattle along with the genes and pathways responsible for it.