Unraveling the Immunogenetics of STAT Proteins: Clinical Perspectives on Gain-of-Function and Loss-of-Function Variants.

Signal Transducer and Activator of Transcription (STAT) proteins play pivotal roles in immune regulation. The dysregulation of these proteins, attributed to both gain-of-function (GOF) and loss-of-function (LOF) variants, has emerged as a substantial and intricate area of research. This comprehensive review delves into the intricate details of the diverse clinical spectrum associated with STAT variants and the immunological findings linked to these genetic alterations. Although this review does not encompass the treatment of each individual disease, we discuss investigative approaches ranging from immunophenotyping assessment to evaluation of STAT protein activity. These investigations play a crucial role in identifying affected patients and understanding the complexities of STAT.

EZH2 as a major histone methyltransferase in PDGF-BB-activated orbital fibroblast in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is an extra-thyroidal complication of Graves' disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system.

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

Association of genetic variation on X chromosome with systemic lupus erythematosus in both Thai and Chinese populations.

OBJECTIVES: X chromosome has been considered as a risk factor for SLE, which is a prototype of autoimmune diseases with a significant sex difference (female:male ratio is around 9:1). Our study aimed at exploring the association of genetic variants in X chromosome and investigating the influence of trisomy X in the development of SLE. METHODS: X chromosome-wide association studies were conducted using data from both Thai (835 patients with SLE and 2995 controls) and Chinese populations (1604 patients with SLE and 3324 controls). Association analyses were performed separately in females and males, followed by a meta-analysis of the sex-specific results. In addition, the dosage of X chromosome in females with SLE were also examined. RESULTS: Our analyses replicated the association of TMEM187-IRAK1-MECP2, TLR7, PRPS2 and GPR173 loci with SLE. We also identified two loci suggestively associated with SLE. In addition, making use of the difference in linkage disequilibrium between Thai and Chinese populations, a synonymous variant in TMEM187 was prioritised as a likely causal variant. This variant located in an active enhancer of immune-related cells, with the risk allele associated with decreased expression level of TMEM187. More importantly, we identified trisomy X (47,XXX) in 5 of 2231 (0.22%) females with SLE. The frequency is significantly higher than that found in the female controls (0.08%; two-sided exact binomial test P=0.002). CONCLUSION: Our study confirmed previous SLE associations in X chromosome, and identified two loci suggestively associated with SLE. More importantly, our study indicated a higher risk of SLE for females with trisomy X.

Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies.

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization.

Efficacy and mechanisms of cannabis oil for alleviating side effects of breast cancer chemotherapy (CBC2): protocol for randomized controlled trial.

BACKGROUND: In a pilot study using both cannabidiol (CBD) and tetrahydrocannabinol (THC) as single agents in advanced cancer patients undergoing palliative care in Thailand, the doses were generally well tolerated, and the outcome measure of total symptom distress scores showed overall symptom benefit. The current study aims to determine the intensity of the symptoms experienced by breast cancer patients, to explore the microbiome profile, cytokines, and bacterial metabolites before and after the treatment with cannabis oil or no cannabis oil, and to study the pharmacokinetics parameters and pharmacogenetics profile of the doses. METHODS: A randomized, double-blinded, placebo-controlled trial will be conducted on the breast cancer cases who were diagnosed with breast cancer and currently receiving chemotherapy at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand. Block randomization will be used to allocate the patients into three groups: Ganja Oil (THC 2 mg/ml; THC 0.08 mg/drop, and CBD 0.02 mg/drop), Metta Osot (THC 81 mg/ml; THC 3 mg/drop), and placebo oil. The Edmonton Symptom Assessment System (ESAS), Food Frequency Questionnaires (FFQ), microbiome profile, cytokines, and bacterial metabolites will be assessed before and after the interventions, along with pharmacokinetic and pharmacogenetic profile of the treatment during the intervention. TRIAL REGISTRATION: TCTR20220809001.

Preservation of Anti-SARS-CoV-2 Neutralizing Antibodies in Breast Milk: Impact of Maternal COVID-19 Vaccination and Infection.

Objectives: To investigate specific immunoglobulin A (sIgA), specific immunoglobulin G (sIgG), and neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in breast milk and compare immunity in mothers with hybrid immunity (infection and vaccination) versus those solely vaccinated (coronavirus disease [COVID]-naïve). Methods: A longitudinal study was conducted among lactating mothers who received at least two doses of the coronavirus disease 2019 (COVID-19) vaccine or tested positive for SARS-CoV-2. Details of vaccination and infection were collected through questionnaires and interviews. Fifteen milliliters of breast milk samples, self-collected at 1, 3, and 6 months postvaccination or infection, were sent to analysis for sIgA, sIgG, and NAbs using enzyme-linked immunosorbent assay. Results: In total, 119 lactating mothers (202 milk samples) were enrolled; 82 participants had hybrid immunity, and 32 were COVID-19-naïve. Two-thirds received a combination of different vaccines and booster shots. Breast milk retained sIgA, sIgG, and NAbs for up to 6 months post-COVID vaccination or infection. At 3 months, mothers with hybrid immunity had significantly higher sIgA and NAbs compared with COVID-naïve mothers (geometric mean [95% confidence interval (CI)] of sIgA 2.72 [1.94-3.8] vs. 1.44 [0.83-2.48]; NAbs 86.83 [84.9-88.8] vs. 81.28 [76.02-86.9]). No differences in sIgA, sIgG, and NAbs were observed between lactating mothers receiving two, three, or more than or equal to three doses, regardless of hybrid immunity or COVID-naïve status. Conclusion: sIgA, sIgG, and NAbs against SARS-CoV-2 in breast milk sustained for up to 6 months postimmunization and infection. Higher immunity was found in mothers with hybrid immunity. These transferred immunities confirm in vitro protection, supporting the safety of breastfeeding during and after COVID-19 vaccination or infection.

H4K20me3 upregulated by reactive oxygen species is associated with tumor progression and poor prognosis in patients with hepatocellular carcinoma.

Epigenetic alteration by oxidative stress is vitally involved in carcinogenesis and cancer progression. Previously, we demonstrated that oxidative stress was increased in hepatocellular carcinoma (HCC) patients and associated with tumor aggressiveness. Herein, we immunohistochemically investigated whether histone methylation, specifically H4K20me3, was upregulated in human hepatic tissues obtained from HCC patients (n = 100). Also, we experimentally explored if the H4K20me3 was upregulated by reactive oxygen species (ROS) and contributed to tumor progression in HCC cell lines. We found that H4K20me3 level was increased in HCC tissues compared with the adjacent noncancerous liver tissues. H3K9me3 and H3K4me3 levels were also increased in HCC tissues. Cox regression analysis revealed that the elevated H4K20me3 level was associated with tumor recurrence and short survival in HCC patients. Experimentally, H2O2 provoked oxidative stress and induced H4K20me3 formation in HepG2 and Huh7 cells. Transcript expression of histone methyltransferase Suv420h2 (for H4K20me3), Suv39h1 (for H3K9me3), and Smyd3 (for H3K4me3) were upregulated in H2O2-treated HCC cells. H2O2 also induced epithelial-mesenchymal transition (EMT) in HCC cells, indicated by decreased E-cadherin but increased α-SMA and MMP-9 mRNA expression. Migration, invasion, and colony formation in HCC cells were markedly increased following the H2O2 exposure. Inhibition of H4K20me3 formation by A196 (a selective inhibitor of Suv420h2) attenuated EMT and reduced tumor migration in H2O2-treated HCC cells. In conclusion, we demonstrated for the first time that H4K20me3 level was increased in human HCC tissues, and it was independently associated with poor prognosis in HCC patients. ROS upregulated H4K20me3 formation, induced mRNA expression of EMT markers, and promoted tumor progression in human HCC cells. Inhibition of H4K20me3 formation reduced EMT and tumor aggressive phenotypes in ROS-treated HCC cells. Possibly, ROS-induced EMT and tumor progression in HCC cells was epigenetically mediated through an increased formation of repressive chromatin H4K20me3.

The Persistence of Specific Immunoglobulin A Against SARS-CoV-2 in Human Milk After Maternal COVID-19 Vaccination.

Objectives: To investigate SARS-CoV-2 specific immunoglobulin A (sIgA) in breast milk of Thai mothers post COVID-19 vaccination and/or SARS-CoV-2 infection, and to compare the sIgA among lactating mothers with varying COVID-19 vaccination regimes. Materials and Methods: A longitudinal study was conducted in lactating mothers receiving ≥2 doses of COVID-19 vaccine or confirming SARS-CoV-2-positive test as a part of an infant feeding survey. Vaccination and infection details were collected through questionnaires and interviews. Self-collected breast milk samples (30 mL) at 1, 3, and 6 months postvaccination or infection were analyzed for sIgA through enzyme-linked immunosorbent assay (ELISA). Results: Eighty-eight lactating mothers (152 milk samples), average age of 30.7 ± 6.2 years, were recruited. Fifty-five percent of milk samples were from lactating mothers with both SARS-CoV-2 infection and vaccination (hybrid immunity); 40% were from those with vaccination alone (COVID naïve). Sixty percent of lactating mothers received mixed types of vaccines. Median sIgA ratio in breast milk was 2.67 (0.82-7.85). Breast milk sIgA at 1, 3, and 6 months were higher in mothers with hybrid immunity than in COVID naïve (geometric mean [95% confidence interval]: 3.30 [2.06-5.29] versus 1.04 [0.52-2.04], 3.39 [2.24-5.13] versus 1.26 [0.77-2.06], 4.29 [3.04-6.06] versus 1.33 [0.74-2.42], respectively). No significant differences were observed among various vaccination regimes. Conclusion: sIgA against SARS-CoV-2 was detected in breast milk for up to 6 months after immunization together with infection at a greater level than after immunization or infection alone. This immunity could be transferred and protective against SARS-CoV-2 infection. Discontinuation of breastfeeding among mothers who received COVID vaccination or experienced infection should be discouraged. Clinical Trial Registration number: TCTR20220215012.

The impact of non-synonymous mutations on miRNA binding sites within the SARS-CoV-2 NSP3 and NSP4 genes.

Non-synonymous mutations in the SARS-CoV-2 spike region affect cell entry, tropism, and immune evasion, while frequent synonymous mutations may modify viral fitness. Host microRNAs, a type of non-coding RNA, play a crucial role in the viral life cycle, influencing viral replication and the host immune response directly or indirectly. Recently, we identified ten miRNAs with a high complementary capacity to target various regions of the SARS-CoV-2 genome. We filtered our candidate miRNAs to those only expressed with documented expression in SARS-CoV-2 target cells, with an additional focus on miRNAs that have been reported in other viral infections. We determined if mutations in the first SARS-CoV-2 variants of concern affected these miRNA binding sites. Out of ten miRNA binding sites, five were negatively impacted by mutations, with three recurrent synonymous mutations present in multiple SARS-CoV-2 lineages with high-frequency NSP3: C3037U and NSP4: G9802U/C9803U. These mutations were predicted to negatively affect the binding ability of miR-197-5p and miR-18b-5p, respectively. In these preliminary findings, using a dual-reporter assay system, we confirmed the ability of these miRNAs in binding to the predicted NSP3 and NSP4 regions and the loss/reduced miRNA bindings due to the recurrent mutations.

Immunogenicity and reactogenicity of fractional, heterologous primary COVID-19 vaccination schedules with BNT162b2 boosters in 5-11-year-old Thai children: A multicenter, prospective, double-blind, randomized control trial.

OBJECTIVE: To evaluate immunogenicity and safety of heterologous COVID-19 primary vaccination regimens of CoronaVac with fractional and standard BNT162b2 dosages in 5-11-year-old Thai children. METHODS: This prospective, multicenter, double-blind, randomized control trial divided participants 1:1:1:1 to receive a second dose of either standard (10-µg) or half-dose (5-µg) BNT162b2 vaccines as follows: CoronaVac/10-µg-BNT162b2 (Group 1), CoronaVac/5-µg-BNT162b2 (Group 2), 10-µg-BNT162b2/10-µg-BNT162b2 (Group 3), or 10-µg-BNT162b2/5-µg-BNT162b2 (Group 4). A subset of participants from each arm received 10-µg-BNT162b2 booster (third) doses 16 weeks after their second vaccination. Humoral and cellular immunogenicity were assessed and adverse events (AEs) digitally self-reported. RESULTS: Of 553 enrolled participants, 50 % were male, the median (interquartile range) age was 8.65 (7.00, 10.00) years, and a majority (91 %) had normal weight-for-height. All participants exhibited similarly robust neutralizing antibodies (NAb) against the ancestral Wuhan strain two weeks after the second vaccination, with titers highest in Group 1 (737.60, 95% CI [654.80, 830.88]), followed by Groups 3 (630.42, 95% CI [555.50, 715.45]), 2 (593.98, 95% CI [506.02, 697.23]), and 4 (451.79, 95% CI [388.62, 525.23]), as well as 56.01 % and 49.68 % seroconversion for BA.1 and BA.5, respectively. Half-dose BNT162b2 as a second dose induced significantly lower NAb titers compared to their respective full-dose regimens (p = 0.03 for Groups 1 vs 2 and p < 0.001 for Groups 3 vs 4). 77.71 % of participants developed SARS-CoV-2 ancestral spike protein-specific T-cell responses two weeks after the second vaccination. This was similar across arms. Booster doses generated NAb titers 5.69-11.51-folds higher than the second vaccination against BA.1. AEs were similar across arms, all mild or moderate, and fully resolved 2-3 days thereafter. CONCLUSION: Standard and fractional heterologous regimens of CoronaVac-BNT162b2 induced similar or higher humoral immunity than homologous BNT162b2 and represent alternative vaccine regimens for children. These findings are highly relevant in settings concurrently using both vaccines.

Ca-EDTA restores the activity of ceftazidime-avibactam or aztreonam against carbapenemase-producing Klebsiellapneumoniae infections.

Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

Attenuated immunogenicity of SARS-CoV-2 vaccines and risk factors in stem cell transplant recipients: a meta-analysis.

Immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is diminished in hematopoietic stem cell transplant (HSCT) recipients. To summarize current evidence and identify risk factors for attenuated responses, 5 electronic databases were searched since database inceptions through 12 January 2023 for studies reporting humoral and/or cellular immunogenicity of SARS-CoV-2 vaccination in the HSCT population. Using descriptive statistics and random-effects models, extracted numbers of responders and pooled odds ratios (pORs) with 95% confidence intervals (CIs) for risk factors of negative immune responses were analyzed (PROSPERO: CRD42021277109). From 61 studies with 5906 HSCT recipients, after 1, 2, and 3 doses of messenger RNA (mRNA) SARS-CoV-2 vaccines, the mean antispike antibody seropositivity rates (95% CI) were 38% (19-62), 81% (77-84), and 80% (75-84); neutralizing antibody seropositivity rates were 52% (40-64), 71% (54-83), and 78% (61-89); and cellular immune response rates were 52% (39-64), 66% (51-79), and 72% (52-86). After 2 vaccine doses, risk factors (pOR; 95% CI) associated with antispike seronegativity were male recipients (0.63; 0.49-0.83), recent rituximab exposure (0.09; 0.03-0.21), haploidentical allografts (0.46; 0.22-0.95), <24 months from HSCT (0.25; 0.07-0.89), lymphopenia (0.18; 0.13-0.24), hypogammaglobulinemia (0.23; 0.10-0.55), concomitant chemotherapy (0.48; 0.29-0.78) and immunosuppression (0.18; 0.13-0.25). Complete remission of underlying hematologic malignancy (2.55; 1.05-6.17) and myeloablative conditioning (1.72; 1.30-2.28) compared with reduced-intensity conditioning were associated with antispike seropositivity. Ongoing immunosuppression (0.31; 0.10-0.99) was associated with poor cellular immunogenicity. In conclusion, attenuated humoral and cellular immune responses to mRNA SARS-CoV-2 vaccination are associated with several risk factors among HSCT recipients. Optimizing individualized vaccination and developing alternative COVID-19 prevention strategies are warranted.

COVID-19 monitoring with sparse sampling of sewered and non-sewered wastewater in urban and rural communities.

Equitable SARS-CoV-2 surveillance in low-resource communities lacking centralized sewers is critical as wastewater-based epidemiology (WBE) progresses. However, large-scale studies on SARS-CoV-2 detection in wastewater from low-and middle-income countries is limited because of economic and technical reasons. In this study, wastewater samples were collected twice a month from 186 urban and rural subdistricts in nine provinces of Thailand mostly having decentralized and non-sewered sanitation infrastructure and analyzed for SARS-CoV-2 RNA variants using allele-specific RT-qPCR. Wastewater SARS-CoV-2 RNA concentration was used to estimate the real-time incidence and time-varying effective reproduction number (Re). Results showed an increase in SARS-CoV-2 RNA concentrations in wastewater from urban and rural areas 14-20 days earlier than infected individuals were officially reported. It also showed that community/food markets were "hot spots" for infected people. This approach offers an opportunity for early detection of transmission surges, allowing preparedness and potentially mitigating significant outbreaks at both spatial and temporal scales.

Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

Less Severe Sepsis in Cecal Ligation and Puncture Models with and without Lipopolysaccharide in Mice with Conditional Ezh2-Deleted Macrophages (LysM-Cre System).

Despite a previous report on less inflammatory responses in mice with an absence of the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, using a lipopolysaccharide (LPS) injection model, proteomic analysis and cecal ligation and puncture (CLP), a sepsis model that more resembles human conditions was devised. As such, analysis of cellular and secreted protein (proteome and secretome) after a single LPS activation and LPS tolerance in macrophages from Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) mice (Ezh2 null) and the littermate control mice (Ezh2fl/fl; LysM-Cre-/-) (Ezh2 control) compared with the unstimulated cells from each group indicated fewer activities in Ezh2 null macrophages, especially by the volcano plot analysis. Indeed, supernatant IL-1ß and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1ß and iNOS), TNF-α, and NF-κB (a transcription factor) were lower in Ezh2 null macrophages compared with the control. In LPS tolerance, downregulated NF-κB compared with the control was also demonstrated in Ezh2 null cells. In CLP sepsis mice, those with CLP alone and CLP at 2 days after twice receiving LPS injection, representing sepsis and sepsis after endotoxemia, respectively, symptoms were less severe in Ezh2 null mice, as indicated by survival analysis and other biomarkers. However, the Ezh2 inhibitor improved survival only in CLP, but not LPS with CLP. In conclusion, an absence of Ezh2 in macrophages resulted in less severe sepsis, and the use of an Ezh2 inhibitor might be beneficial in sepsis.

Immunosuppressive Polymeric Nanoparticles Targeting Dendritic Cells Alleviate Lupus Disease in Fcgr2b-/- Mice by Mediating Antigen-Specific Immune Tolerance.

Dendritic cells (DCs) are the most potent antigen-presenting cells that have multifaceted functions in the control of immune activation and tolerance. Hyperresponsiveness and altered tolerogenicity of DCs contribute to the development and pathogenesis of system lupus erythematosus (SLE); therefore, DC-targeted therapies aimed at inducing specific immune tolerance have become of great importance for the treatment of SLE. This study developed a new nanoparticle (NP) containing a biodegradable PDMAEMA-PLGA copolymer for target-oriented delivery to DCs in situ. PDMAEMA-PLGA NPs provided sustained drug release and exhibited immunosuppressive activity in FLT3L and GM-CSF-derived bone marrow in conventional DCs (BM-cDCs). PDMAEMA-PLGA NPs improved dexamethasone capability to convert wild-type and Fcgr2b-/- BM-cDCs from an immunogenic to tolerogenic state, and BM-cDCs treated with dexamethasone-incorporated PDMAEMA-PLGA NPs (Dex-NPs) efficiently mediated regulatory T cell (Treg) expansion in vitro. Dex-NP therapy potentially alleviated lupus disease in Fcgr2b-/- mice by mediating Foxp3+ Treg expansion in an antigen-specific manner. Our findings substantiate the superior efficacy of DC-targeted therapy using the PDMAEMA-PLGA NP delivery system and provide further support for clinical development as a potential therapy for SLE. Furthermore, PDMAEMA-PLGA NP may be a versatile platform for DC-targeted therapy to induce antigen-specific immune tolerance to unwanted immune responses that occur in autoimmune disease, allergy, and transplant rejection.

Human gut, breast, and oral microbiome in breast cancer: A systematic review and meta-analysis.

Introduction: Dysbiosis characterises breast cancer through direct or indirect interference in a variety of biological pathways; therefore, specific microbial patterns and diversity may be a biomarker for the diagnosis and prognosis of breast cancer. However, there is still much to determine about the complex interplay of the gut microbiome and breast cancer. Objective: This study aims to evaluate microbial alteration in breast cancer patients compared with control subjects, to explore intestine microbial modification from a range of different breast cancer treatments, and to identify the impact of microbiome patterns on the same treatment-receiving breast cancer patients. Methods: A literature search was conducted using electronic databases such as PubMed, Embase, and the CENTRAL databases up to April 2021. The search was limited to adult women with breast cancer and the English language. The results were synthesised qualitatively and quantitatively using random-effects meta-analysis. Results: A total of 33 articles from 32 studies were included in the review, representing 19 case-control, eight cohorts, and five nonrandomised intervention researches. The gut and breast bacterial species were elevated in the cases of breast tumours, a significant increase in Methylobacterium radiotolerans (p = 0.015), in compared with healthy breast tissue. Meta-analysis of different α-diversity indexes such as Shannon index (p = 0.0005), observed species (p = 0.006), and faint's phylogenetic diversity (p < 0.00001) revealed the low intestinal microbial diversity in patients with breast cancer. The microbiota abundance pattern was identified in different sample types, detection methods, menopausal status, nationality, obesity, sleep quality, and several interventions using qualitative analysis. Conclusions: This systematic review elucidates the complex network of the microbiome, breast cancer, and therapeutic options, with the objective of providing a link for stronger research studies and towards personalised medicine to improve their quality of life.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Effect of Probiotics in Breast Cancer: A Systematic Review and Meta-Analysis.

Probiotics may have the potential to protect against breast cancer, partly through systemic immunomodulatory action and active impact upon intestinal microbiota. Given a few clinical studies on their curative role, we conducted a systematic review of the potential effects of probiotics in breast cancer patients and survivors of breast cancer, aiming to support further clinical studies. A literature search was performed using PubMed, Embase, and the CENTRAL databases from inception through to March 2022. A total of eight randomized clinical trials were identified from thirteen articles published between 2004 and 2022. We evaluated quality-of-life measures, observed bacterial species and diversity indices, probiotic-related metabolites, inflammatory biomarkers, and other responses in breast cancer patients and survivors. Results were synthesized qualitatively and quantitatively using random-effects meta-analysis. Different probiotics supplements utilized included Lactobacillus species alone (Lacto), with or without estriol; probiotic combinations of Lactobacillus with Bifidobacterium (ProLB), with or without prebiotic fructooligosaccharides (FOS); ProLB plus Streptococcus and FOS (ProLBS + FOS); and ProLB plus Enterococcus (ProLBE). We found that use of ProLBS with FOS in breast cancer patients and use of ProLBE in survivors of breast cancer show potential benefits in countering obesity and dyslipidemia. ProLBS with FOS use decreases pro-inflammatory TNF-α in breast cancer survivors and improves quality of life in those with breast-cancer-associated lymphedema. Supplementing probiotics capsules (109 CFU) with a prebiotic and using an intake duration of 10 weeks could provide a better approach than probiotics alone.