Monocytic-Myeloid Derived Suppressor Cells Suppress T-Cell Responses in Recovered SARS CoV2-Infected Individuals.

Coronavirus disease 2019 (COVID-19) caused by SARS Coronavirus 2 (CoV2) is associated with massive immune activation and hyperinflammatory response. Acute and severe CoV2 infection is characterized by the expansion of myeloid derived suppressor cells (MDSC) because of cytokine storm, these MDSC suppress T cell functions. However, the presence of MDSC and its effect on CoV2 antigen specific T cell responses in individuals long after first detection of CoV2 and recovery from infection has not been studied. We and others have previously shown that CD11b+CD33+CD14+HLA-DR-/lo monocytic MDSC (M-MDSC) are present in individuals with clinical recovery from viral infection. In this study, we compared the frequency, functional and transcriptional signatures of M-MDSC isolated from CoV2 infected individuals after 5-months of the first detection of the virus (CoV2+) and who were not infected with CoV2 (CoV2-). Compared to CoV2- individuals, M-MDSC were present in CoV2+ individuals at a higher frequency, the level of M-MDSC correlated with the quantity of IL-6 in the plasma. Compared to CoV2-, increased frequency of PD1+, CD57+ and CX3CR1+ T effector memory (TEM) cell subsets was also present in CoV2+ individuals, but these did not correlate with M-MDSC levels. Furthermore, depleting M-MDSC from peripheral blood mononuclear cells (PBMC) increased T cell cytokine production when cultured with the peptide pools of immune dominant spike glycoprotein (S), membrane (M), and nucleocapsid (N) antigens of CoV2. M-MDSC suppressed CoV2 S- antigen-specific T cell in ROS, Arginase, and TGFß dependent manner. Our gene expression, RNA-seq and pathway analysis studies further confirm that M-MDSC isolated from CoV2+ individuals are enriched in pathways that regulate both innate and adaptive immune responses, but the genes regulating these functions (HLA-DQA1, HLA-DQB1, HLA-B, NLRP3, IL1ß, CXCL2, CXCL1) remained downregulated in M-MDSC isolated from CoV2+ individuals. These results demonstrate that M-MDSC suppresses recall responses to CoV2 antigens long after recovery from infection. Our findings suggest M-MDSC as novel regulators of CoV2 specific T cell responses, and should be considered as target to augment responses to vaccine.

Global DNA hypomethylation and the expression profile of DNA methyltransferase genes in late-onset neonatal sepsis.

Aim: Infectious organisms tend to cause DNA methylation changes. Thus, this paper aims to study global DNA methylation and the expression of DNA methyltransferase (DNMT) genes in late-onset neonatal sepsis (LONS). Methods: Global and Alu DNA methylation and expression levels of DNMT were performed using 5mc ELISA, methylation-specific PCR and quantitative real-time-PCR, respectively for LONS and controls. Results: Significant hypomethylation of global DNA and Alu DNA methylation and lower expression of DNMT1 and DNMT3a were observed in LONS compared with controls. Receiver operating characteristic analysis of global and Alu DNA methylation showed good discrimination for the identification of LONS. Conclusion: The hypomethylation of global DNA and Alu elements is evident in neonates with LONS. This may be clinically useful for the prognosis of LONS.

MicroRNA Expression in Neonates with Late-onset Sepsis - A Cross-sectional Comparative Study.

BACKGROUND: Neonatal sepsis is a major health concern among neonates with higher morbidity and mortality rate. Studies have recently speculated the importance of differential expression of circulating mature micro-RNAs (miRNAs) which could serve as diagnostic as well as prognostic markers for risk of mortality in neonatal sepsis. This study aimed to analyze the expression pattern and to assess the diagnostic/prognostic value of miRNAs miR-21, miR-29a miR-31, miR-146a, and miR-155 in late-onset neonatal sepsis. METHODS: A cross-sectional comparative study was conducted including 42 healthy controls and 42 neonates with late-onset neonatal sepsis. SYBR green-based miScript RT-PCR assay was used for the expression analysis and the comparative Ct method 2-delta (Ct) method was used for relative quantification of the candidate miRNAs in plasma. Significantly higher expression of miR-21 and miR-155 and lower expression of miR-29a and miR-146a was observed in cases compared to control except miR-31. In subgroups analysis, miR-21(p = .03) showed a significant difference between pre-term and term neonates and miR-31 (p = .01) and miR-155 (p = .03) showed a significant difference between low-birth-weight and normal-birth-weight neonates. miR-146a showed significantly lower expression in the non-survivor group compared to the survivor group (p = .005). A receiver operating characteristic curve (ROC) analysis of miR-21 and miR-29a (0.829 and 0.787 AUC of ROC curves) showed good discrimination for the identification of sepsis from non-sepsis neonates. CONCLUSION: The current study shows evidence of differential expression of miRNAs in neonatal sepsis and this altered expression of candidate miRNAs could be involved in immune dysregulation, thus leading to sepsis-related severity in newborns.

Methylation Status of VDR Gene and its Association with Vitamin D Status and VDR Gene Expression in Pediatric Tuberculosis Disease.

Deficiency in circulatory vitamin D level and vitamin D receptor DNA methylation could be associated with weakened innate immune response and increased susceptibility to tuberculosis (TB) disease in children. Therefore, we aimed to study the effect of vitamin D receptor (VDR) gene methylation on plasma vitamin D level and the expression of the VDR gene in children with active-TB disease. A cross-sectional comparative study was conducted in 43 children with active-TB and 33 healthy control children (HC). The vitamin D level was measured in plasma, while the levels of VDR gene promoter methylation and VDR gene expression were measured in peripheral blood. Children with active-TB showed a significantly lower median vitamin D level than HC [Cases 17.18 ng/mL (IQR, 8.3-18.6 ng/mL); HC 41.34 ng/mL (IQR, 40.2-43.49 ng/mL) (p<0.0001)] and decreased mRNA expression level of VDR gene [Cases 0.51 (IQR, 0.40-0.70); HC 1.06 (IQR, 0.8-1.2) (p<0.0001)] and increased VDR DNA methylation [Cases 75% (IQR, 50-75%); HC 10% (IQR, 10-25%) (p<0.0001)]. The VDR hypermethylation is significantly associated with reduced vitamin D level and decreased expression level of VDR gene. Therefore this inverse association could be involved in the impairment in the VDR mediated cytolytic and antimicrobial effector cell response in pediatric TB disease.

Assessment of global DNA methylation in children with tuberculosis disease.

Background: Studying DNA methylation changes in disease condition provides the basis of disease pathogenesis or host immune response to infection. Evidences suggest that pathogen-mediated DNA methylation changes influences the expression pattern of genes contributing to disease condition to avert host immune response. Hence, we attempted to study the association between tubercle bacilli-mediated global DNA methylation changes in children with tuberculosis (TB) disease and healthy controls. Methods: Forty-three children diagnosed with TB and 33 healthy children were enrolled in this study. ELISA-based global DNA methylation quantification was performed to measure the changes in percentage of global genomic DNA methylation level. Results: Highly significant difference in global DNA methylation level was found between cases and controls and median global DNA methylation level was 6.25% (interquartile range [IQR] 2.5%-10%) in cases and 25% (IQR, 12.5%-25%) in controls (P < 0.001). Significant difference was found in GeneXpert-positive cases (P < 0.01). Receiver operating curve analysis showed that area under curve 0.81 for the total study population and 0.76 for GeneXpert-positive cases. Conclusion: The results show the significant difference in global DNA methylation level in children with TB disease that can serve as a potential biomarker in early diagnosis of TB disease. Measuring global DNA methylation level, however, not an accurate or sensitive diagnostic method but evaluating active demethylation at genome-wide level can be used to monitor disease progression and treatment efficacy.

Methylation status of alu repetitive elements in children with tuberculosis disease.

Background: Investigation of DNA methylation in Alu repetitive elements (REs) was shown to be a promising field to explore transcriptional changes in human genome under disease condition. To scrutinize the association between Alu methylation and tuberculosis (TB) disease in children, the difference in Alu DNA methylation level was compared with healthy controls. Methods: Whole-blood genomic DNA from 36 TB-infected children and 32 healthy controls was isolated, and the level of Alu repeat DNA methylation was examined by methylation-specific polymerase chain reaction. Results: The median Alu methylation level in TB patients was 30% (Interquartile range [IQR], 25-30%), whereas in healthy controls, it was 75% (IQR, 50-75%) (P < 0.0001). The median level of DNA methylation of Alu RE in TB cases was significantly lower than healthy controls. Receiver operating characteristic curve analysis showed that the area under the curve for diagnosis was 0.969 (95% confidence interval, 0.936-1) (P < 0.0001), with 100% sensitivity and 84% specificity. Conclusion: Our results point out that detection of Alu DNA methylation in whole-blood DNA may be clinically useful tool for the diagnosis and prognosis of TB disease in children.

Molecular typing and differentiation of Mycobacterium tuberculosis clinical isolates using Double Repetitive Element PCR and Duplex PCR.

BACKGROUND: To date, the advancements in polymerase chain reaction (PCR) assures accurate, fast identification and mycobacterial speciation in clinical settings, which promotes a better tuberculosis (TB) treatment regimen. METHODS: In this study, a total of 78 clinically suspected cases of TB were processed for the detection of Mycobacterial infections by standard Ziehl Neelsen (ZN) staining, conventional Lowenstein-Jensen (LJ) and BACTEC MGIT-960™ liquid culture. Strain typing was performed by using Double Repetitive Element PCR (DRE-PCR) and Duplex PCR (DPCR) to differentiate Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM), respectively. RESULTS: Of 78 clinical isolates, 25 (32%) were drug-susceptible, and 53 (68%) were resistant to at least one drug. The BACTEC MGIT-960™ showed the highest (88.5%) positivity rate, compared with conventional LJ (82%) and ZN smear (61.5%). The mean time detection and drug susceptibility for MTB was 28 and 40days in LJ culture, and 10 and 13 days in BACTEC MGIT-960™ culture. Using DPCR, Mycobacterium avium infection was identified in HIV-positive (2.56%) and MTB in HIV-negative patients (97.4%), and the DRE-PCR system divulged 15 unique genotype patterns, and an institutional-based epidemiology database was created. CONCLUSIONS: The combination of an in-house DRE-DPCR system could possibly identify and differentiate MTB from other mycobacterial species in a single reaction. In addition, restriction polymorphism analysis and DNA sequencing of NTM could assist in species identification directly from clinical isolates.