Peripheral Leukocytes Research Articles

Orchestration of a blood-derived and ADARB1-centred network in Alzheimer's and Parkinson's disease

The peripheral immune system is thought to influence the pathogenesis of the central nervous system in Alzheimer's disease (AD) and Parkinson's disease (PD). This study aimed to investigate the characteristics of peripheral leukocytes in AD and PD by comprehensively analyzing the transcriptomic and metabolic features in the blood (NCONTROL = 15; NAD = 11; NPD = 13). The study found an ADARB1-centered module that was associated with diagnosis, phenethylamine, and glutamate. The module consisted of ADARB1, a vital RNA-editing enzyme, LINC02960, and 109 miRNAs. The study also predicted that the ADARB1 and involved regulators were targeted by miRNAs in the ADARB1 module. The integrated analysis of transcriptome and metabolic panel revealed a central role of ADARB1, miR-199b-5p, miR-26a, miR-450b-5p, miR-34c-5p, glutamate and phenethylamine in the regulatory relationships. The study highlights a set of synergetic non-coding RNA related to ADARB1 in the blood ecosystem of AD and PD with dynamic glutamate and phenethylamine, providing new insights into the pathogenesis of these diseases.

Components of the Glutathione Cycle as Markers of Biological Age: An Approach to Clinical Application in Aging.

The oxidative-inflammatory theory of aging states that aging is the result of the establishment of a chronic oxidative-inflammatory stress situation in which the immune system is implicated. Among the redox parameters, those involved in the glutathione cycle have been suggested as essential in aging. Thus, the first objective of this study was to determine if several components of the glutathione cycle (glutathione reductase (GR) and glutathione peroxidase (GPx) activities, and concentrations of oxidized glutathione (GSSG) and reduced glutathione (GSH)) in leukocytes) are associated with the biological age (ImmunolAge) estimated using the Immunity Clock in 190 men and women. The second objective was to identify the best blood fraction (whole blood, blood cells, erythrocytes, or plasma) to quantify these components and correlate them with the estimated ImmunolAge. The results show that the oxidative state of peripheral leukocytes correlates with their functionality, supporting the idea that this is the basis of immunosenescence. In blood, the correlations are more significant in the fraction of blood cells with respect to ImmunolAge (positive correlations with GSSG concentration and the GSSG/GSH ratio, and negative correlations with GPx and GR activities). Therefore, blood cells are proposed as the most effective sample to estimate the biological age of individuals in clinical settings.

Circulating macrophages as the mechanistic link between mosaic loss of Y-chromosome and cardiac disease

BackgroundGenetics evidences have long linked mosaic loss of Y-chromosome (mLOY) in peripheral leukocytes with a wide range of male age-associated diseases. However, a lack of cellular and molecular mechanistic explanations for this link has limited further investigation into the relationship between mLOY and male age-related disease. Excitingly, Sano et al. have provided the first piece of evidence directly linking mLOY to cardiac fibrosis through mLOY enriched profibrotic transforming growth factor β1 (TGF-β1) regulons in hematopoietic macrophages along with suppressed interleukin-1β (IL-1β) proinflammatory regulons. The results of this novel finding can be extrapolated to other disease related to mLOY, such as cancer, cardiac disease, and age-related macular degeneration.ResultsSano et al. used a CRISPR-Cas9 gRNAs gene editing induced Y-chromosome ablation mouse model to assess results of a UK biobank prospective analysis implicating the Y-chromosome in male age-related disease. Using this in vivo model, Sano et al. showed that hematopoietic mLOY accelerated cardiac fibrosis and heart failure in male mice through profibrotic pathways. This process was linked to monocyte-macrophage differentiation during hematopoietic development. Mice confirmed to have mLOY in leukocytes, by loss of Y-chromosome genes Kdm5d, Uty, Eif2s3y, and Ddx3y, at similar percentages to the human population were shown to have accelerated rates of interstitial and perivascular fibrosis and abnormal echocardiograms. These mice also recovered poorly from the transverse aortic constriction (TAC) model of heart failure and developed left ventricular dysfunction at higher rates. This was attributed to aberrant proliferation of cardiac MEF-SK4 + fibroblasts promoted by mLOY macrophages enriched in profibrotic regulons and lacking in proinflammatory regulons. These pro-fibrotic macrophages localized to heart and eventually resulted in cardiac fibrosis via enhanced TGF-β1 and suppressed IL-1β signaling. Furthermore, treatment of mLOY mice with TGFβ1 neutralizing antibody was able to improve their cardiac function. This study by Sano et al. was able to provide a causative link between the known association between mLOY and male cardiac disease morbidity and mortality for the first time, and thereby provide a new target for improving human health.ConclusionsUsing a CRISPR-Cas9 induced Y-chromosome ablation mouse model, Sano et al. has proven mosaic loss of Y-chromosome in peripheral myeloid cells to have a causative effect on male mobility and mortality due to male age-related cardiac disease. They traced the mechanism of this effect to hyper-expression of the profibrotic TGF-β1 and reduced pro-inflammatory IL-1β signaling, attenuation of which could provide another potential strategy in improving outcomes against age-related diseases in men.

Benchmark Dose Assessment for Coke Oven Emissions-Induced Mitochondrial DNA Copy Number Damage Effects*

ObjectiveThe study aimed to estimate the benchmark dose (BMD) of coke oven emissions (COEs) exposure based on mitochondrial damage with the mitochondrial DNA copy number (mtDNAcn) as a biomarker. MethodsA total of 782 subjects were recruited, including 238 controls and 544 exposed workers. The mtDNAcn of peripheral leukocytes was detected through the real-time fluorescence-based quantitative polymerase chain reaction. Three BMD approaches were used to calculate the BMD of COEs exposure based on the mitochondrial damage and its 95% confidence lower limit (BMDL). ResultsThe mtDNAcn of the exposure group was lower than that of the control group (0.60 ± 0.29 vs. 1.03 ± 0.31; P < 0.001). A dose–response relationship was shown between the mtDNAcn damage and COEs. Using the Benchmark Dose Software, the occupational exposure limits (OELs) for COEs exposure in males was 0.00190 mg/m3. The OELs for COEs exposure using the BBMD were 0.00170 mg/m3 for the total population, 0.00158 mg/m3 for males, and 0.00174 mg/m3 for females. In possible risk obtained from animal studies (PROAST), the OELs of the total population, males, and females were 0.00184, 0.00178, and 0.00192 mg/m3 respectively. ConclusionBased on our conservative estimate, the BMDL of mitochondrial damage caused by COEs is 0.002 mg/m3. This value will provide a benchmark for determining possible OELs.

TNF-α-1031T/C gene polymorphism as a predictor of malnutrition in patients with gastric cancer.

Malnutrition is a complex clinical syndrome, the exact mechanism of which is yet not fully understood. Studies have found that malnutrition is associated with anorexia and inadequate intake, tumor depletion, leptin, tumor-induced metabolic abnormalities in the body, and catabolic factors produced by the tumor in the circulation and cytokines produced by the host immune system. Among these, single nucleotide polymorphisms (SNPs) are present in the gene encoding the pro-inflammatory cytokine TNF-α. The objective of this study was to investigate TNF-α -1,031 T/C gene polymorphism as an unfavorable predictor of malnutrition in patients with gastric cancer. The study group consisted of 220 gastric cancer patients treated at Affiliated Jinhua Hospital, Zhejiang University School of Medicine. Malnutrition was mainly assessed by the Global Consensus on Malnutrition Diagnostic Criteria (GLIM). DNA was extracted from peripheral leukocytes of whole blood samples using an animal DNA extraction kit. DNA was amplified using a 1.1× T3 Super PCR mixture and genotyped using SNP1 software. There are three major genetic polymorphisms in TNF-α. Among the 220 patients with gastric cancer, there were 7 patients with the CC genotype, 61 with the CT genotype and 152 with the TT genotype. Compared to patients with the TT genotype, patients with the C allele had an approximately 2.5-fold higher risk of developing malnutrition (p = 0.003; OR = 0.406). On the basis of multivariate analysis, patients with the CC genotype had an approximately 20.1-fold higher risk of developing malnutrition (p = 0.013; OR = 20.114), while those with the CT genotype had an almost 3.7-fold higher risk of malnutrition (p = 0.002; OR = 3.218). SNP (-1,031 T/C) of the TNF-α may be a useful marker in the assessment of the risk of nutritional deficiencies in gastric cancer patients. Patients with gastric cancer carrying the C allele should be supported by early nutritional intervention, but more research is still needed to explore confirmation.

Differential gene expression in peripheral leukocytes of pre-weaned dairy calves with respiratory disease

Bovine respiratory disease (BRD) is a leading cause of calf morbidity and mortality in both beef and dairy sectors of the United States cattle industry resulting in financial losses and compromised welfare. Differential gene expression (DGE) al­lows detailed insight into individual immune responses and can identify enriched pathways and biomarkers that contrib­ute to disease susceptibility and outcome. Differential expres­sion of genes related to innate immune function has been well documented in both feedlot and post-weaned dairy cattle with experimentally induced or naturally occurring BRD. However, little is known regarding DGE in pre-weaned Holstein calves. Therefore, the aims of this study were to evaluate gene expres­sion in peripheral leukocytes relative to BRD diagnosis, dis­ease progression and age progression in Holstein heifer calves throughout the preweaning period.

Comparison of Telomere Length in Age-Matched Primiparous and Multiparous Brahman Cows.

Physiological and psychological stressors have been associated with the attrition of telomeres, which are the protective caps of chromosomes. This study compares the telomere length (TL) in 4-year-old Brahman cows grouped by the first parity (n = 8) and the second parity (n = 11). The cows were bled via jugular venipuncture, weighed, and had their body condition scores recorded at Day -28 prior to calving and at Day + 7 and Day + 28 post-calving. The duration of labor (Dlabor) and parturition ease were recorded. The peripheral leukocytes were isolated, the leukocyte blood count with differential was recorded, and the genomic DNA was extracted. The relative quantity of telomere products, which is proportional to the average TL, was determined via multiplex quantitative PCR using the ratio (T/S ratio) of bovine telomere and β-globulin DNA. Standards of the bovine telomere (1012-107 dilution series) and β-globulin (109-104 dilution series) genes were utilized to produce relative copy numbers. The samples were assayed in triplicate and were included if the triplicate Cq difference was less than 0.25 cycles. The parity was the fixed effect, and the random effects included the sire and day repeated with the cow as the subject. Statistical significance was not observed in the leukocyte number or type (p > 0.1). A reduction in the TL of approximately 9225 telomeric copies was found between Parity 1 and Parity 2 (p = 0.02). A trend was found between the TL and Dlabor (p = 0.06). The stress of parturition and raising the first calf of a cow's life may be responsible for TL attenuation. Parity may be considered a stressor of cow longevity.

Genome-wide analysis of DNA methylation and its relationship with serum homocysteine levels in patients with hypertension.

Homocysteine (Hcy) is an independent risk factor for cardiovascular diseases, and elevated plasma Hcy levels could aggravate vascular injury in hypertension. Hyperhomocysteinemia can change the methylation status of global DNA and specific genes. In the present study, we aim to examine the comprehensive influence of Hcy levels on DNA methylation status in patients with hypertension. Epigenome-wide methylation profiles of the peripheral leukocyte DNA of 218 patients with hypertension were analyzed using the Illumina Infinium Methylation EPIC BeadChip. Differentially methylated positions (DMPs) associated with serum Hcy levels were identified by mixed linear regression with the adjustment of potential confounders. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted to determine the potential functions of the identified DMPs. The association between the methylation level of DMPs and carotid-femoral pulse wave velocity (Cf-PWV) was also analyzed. Five DMPs at cg13169662, cg03179312, cg21976560, cg25262698, and cg09433843 showed significant association with serum Hcy levels (false discovery rate-corrected P < 0.05). An additional six CpG sites met the threshold for suggestive significance ( P < 1 × 10 -6 ), among which three DMPs (cg25781123, cg26463106, and cg06679221) were annotated to THUMPD3 . Furthermore, the methylation levels of cg13169662 and cg25262698 (RPRD1A) were significantly associated with Cf-PWV. Our results suggest that Hcy could induce DNA methylation alteration in patients with hypertension. Further functional research is warranted to elucidate the concrete role of DMPs in hypertension.

Derivation of embryonic stem cells from wild-derived mouse strains by nuclear transfer using peripheral blood cells

Wild-derived mouse strains have been extensively used in biomedical research because of the high level of inter-strain polymorphisms and phenotypic variations. However, they often show poor reproductive performance and are difficult to maintain by conventional in vitro fertilization and embryo transfer. In this study, we examined the technical feasibility of derivation of nuclear transfer embryonic stem cells (ntESCs) from wild-derived mouse strains for their safe genetic preservation. We used leukocytes collected from peripheral blood as nuclear donors without sacrificing them. We successfully established 24 ntESC lines from two wild-derived strains of CAST/Ei and CASP/1Nga (11 and 13 lines, respectively), both belonging to Mus musculus castaneus, a subspecies of laboratory mouse. Most (23/24) of these lines had normal karyotype, and all lines examined showed teratoma formation ability (4 lines) and pluripotent marker gene expression (8 lines). Two male lines examined (one from each strain) were proven to be competent to produce chimeric mice following injection into host embryos. By natural mating of these chimeric mice, the CAST/Ei male line was confirmed to have germline transmission ability. Our results demonstrate that inter-subspecific ntESCs derived from peripheral leukocytes could provide an alternative strategy for preserving invaluable genetic resources of wild-derived mouse strains.

Mitochondrial DNA copy number in autism spectrum disorder and attention deficit hyperactivity disorder: a systematic review and meta-analysis.

Several reports suggest that altered mitochondrial DNA copy number (mtDNA-cn), a common biomarker for aberrant mitochondrial function, is implicated in autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD), but the results are still elusive. A meta-analysis was performed to summarize the current indication and to provide a more precise assessment of the mtDNA-cn in ASD and ADHD. A search in the MEDLINE-PubMed, Scopus, and EMBASE databases was done to identify related studies up to the end of February 2023. The meta-analysis was conducted according to recommendations of the Cochrane Handbook of Systematic Reviews. Fourteen studies involving 666 cases with ASD and ADHD and 585 controls were collected and judged relevant for the systematic review and meta-analysis. The pooled results by a random effects meta-analysis was reported as a geometric mean of the estimated average response ratio and 95% confidence interval. Overall analysis of studies reported differences in mtDNA-cn in blood samples (k = 10) and non-blood samples (brain tissues and oral samples; k = 4) suggested significantly higher mtDNA-cn in patients compared to controls (p = 0.0275). Sub-analysis by stratifying studies based on tissue type, showed no significant increase in mtDNA-cn in blood samples among patients and controls (p = 0.284). Conversely, higher mtDNA-cn was observed in non-blood samples in patients than in controls (p = 0.0122). Further stratified analysis based on blood-cell compositions as potential confounds showed no significant difference in mtDNA-cn in peripheral blood samples of patients comparted to controls (p = 0.074). In addition, stratified analysis of aged-matched ASD and ADHD patients and controls revealed no significant difference in mtDNA-cn in blood samples between patients and controls (p = 0.214), whereas a significant increase in mtDNA-cn was observed in non-blood samples between patients and controls (p < 0.001). Finally, when the mtDNA-cn was analyzed in blood samples of aged-matched patients with ASD (peripheral blood, leukocytes, and PBMCs) or ADHD (peripheral blood), no significant difference in mtDNA-cn was observed between ASD patients and controls (p = 0.385), while a significant increase in mtDNA-cn was found between ADHD patients and controls (p = 0.033). In this first meta-analysis of the evaluation of mtDNA-cn in ASD/ADHD, our results show elevated mtDNA-cn in ASD and ADHD, further emphasizing the implication of mitochondrial dysfunction in neurodevelopmental disorders. However, our results indicate that the mtDNA-cn in blood is not reflected in other tissues in ASD/ADHD, and the true relationship between blood-derived mtDNA-cn and ASD/ADHD remains to be defined in future studies. The importance of blood-cell compositions as confounders of blood-based mtDNA-cn measurement and the advantages of salivary mtDNA-cn should be considered in future studies. Moreover, the potential of mtDNA-cn as a biomarker for mitochondrial malfunction in neurodevelopmental disorders deserves further investigations.

Peptides Derived from Soybean β-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes.

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein β-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of β-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean β-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

A phase Ib trial of pembrolizumab plus paclitaxel or flat-dose capecitabine in 1st/2nd line metastatic triple-negative breast cancer

Chemoimmunotherapy with anti-programmed cell death 1/ligand 1 and cytotoxic chemotherapy is a promising therapeutic modality for women with triple-negative breast cancer, but questions remain regarding optimal chemotherapy backbone and biomarkers for patient selection. We report final outcomes from a phase Ib trial evaluating pembrolizumab (200 mg IV every 3 weeks) with either weekly paclitaxel (80 mg/m2 weekly) or flat-dose capecitabine (2000 mg orally twice daily for 7 days of every 14-day cycle) in the 1st/2nd line setting. The primary endpoint is safety (receipt of 2 cycles without grade III/IV toxicities requiring discontinuation or ≥21-day delays). The secondary endpoint is efficacy (week 12 objective response). Exploratory aims are to characterize immunologic effects of treatment over time, and to evaluate novel biomarkers. The trial demonstrates that both regimens meet the pre-specified safety endpoint (paclitaxel: 87%; capecitabine: 100%). Objective response rate is 29% for pembrolizumab/paclitaxel (n = 4/13, 95% CI: 10–61%) and 43% for pembrolizumab/capecitabine (n = 6/14, 95% CI: 18–71%). Partial responses are observed in two subjects with chemo-refractory metaplastic carcinoma (both in capecitabine arm). Both regimens are associated with significant peripheral leukocyte contraction over time. Response is associated with clinical PD-L1 score, non-receipt of prior chemotherapy, and the H&E stromal tumor-infiltrating lymphocyte score, but also by a novel 27 gene IO score and spatial biomarkers (lymphocyte spatial skewness). In conclusion, pembrolizumab with paclitaxel or capecitabine is safe and clinically active. Both regimens are lymphodepleting, highlighting the competing immunostimulatory versus lymphotoxic effects of cytotoxic chemotherapy. Further exploration of the IO score and spatial TIL biomarkers is warranted. The clinical trial registration is NCT02734290.

Impact of peripheral mitochondrial DNA level on immune response after COVID-19 vaccination

The efficacy of vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the elderly is partially hindered by immunosenescence, resulting from decreased mtDNA levels. This study evaluated the correlation between mtDNA levels in peripheral leukocytes and immune response to the SARS-CoV-2 vaccine. Two hundred ten participants (median age 79.5 years), including 83 frail residents/inpatients and 70 robust outpatients, were analyzed. Anti-spike IgG antibody (IgG(S)) titers were serially measured from before the first vaccination to after the third vaccination. The mtDNA levels and cell-mediated immunity were measured in 45 elderly and 22 elderly individuals two months after the third vaccination. The robust group had consistently higher IgG(S) titers than the frail group. The mtDNA levels positively correlated with IgG(S) titers, as well as with cell-mediated immunity. These findings suggest that mtDNA levels positively impact vaccine-induced immunity. Further studies into maintaining mtDNA levels may provide insights into immunosenescence in the elderly.

GENOME-WIDE DNA METHYLATION ANALYSIS IDENTIFIES SERUM HOMOCYSTEINE-ASSOCIATED CPG SITES IN HYPERTENSIVES

Objective: Homocysteine (Hcy) is associated with DNA methylation in cardiovascular disease (CVD). Moreover, the relationship between Hyperhomocysteine (HHcy) and CVD risk is stronger in individuals with hypertension. However, no research has focused on the influence of Hcy on DNA methylation status in patients with hypertension. We performed an epigenome-wide association study to identify DNA methylation loci associated with Hcy among 218 hypertensive patients using the Illumina Infinium Methylation EPIC BeadChip (850k chip) and further explored their effects on arteriosclerosis. Design and method: Epigenome-wide methylation profiles were assessed using the Illumina Infinium Methylation EPIC BeadChip (850k chip) in peripheral leukocyte DNA from 218 patients with hypertension. Differentially methylated positions (DMPs) associated with serum homocysteine levels were identified using a mixed linear regression with adjustment for potential confounders. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted to explore the possible functions of the identified DMPs. The association of these differentially methylated loci with hypertension- and Hcy- related arteriosclerosis, Cf-PWV (Carotid-femoral pulse wave velocity) was further analyzed. Results: Five DMPs at cg13169662, cg03179312, cg21976560, cg25262698, and cg09433843 showed significant association with serum Hcy levels (FDR-corrected p &lt;0.05). Six additional CpG sites met the threshold for suggestive significance (p&lt;1 × 10-6), among which three DMPs (cg25781123, cg26463106, and cg06679221) were annotated to the THUMPD3 gene. Furthermore, the methylation levels of cg13169662 and cg25262698 (RPRD1A) were significantly associated with Cf-PWV. Conclusions: Our study provides preliminary evidence for an association between DNA methylation and serum Hcy levels in patients with hypertension. This study may give an insight into the role of DNA methylation in cardiovascular injury underlying HHcy in patients with hypertension.

Differential expression and correlation of immunoregulation related piRNA in rheumatoid arthritis.

Although PIWI-interacting RNAs (piRNAs) have recently been associated with germline development and many human diseases, their expression pattern and relationship in autoimmune diseases remain indistinct. This study aimed to investigate the presence and correlation of piRNAs in rheumatoid arthritis (RA). We first analyzed the expression profile of piRNAs using small RNA sequencing in peripheral leukocytes of three new-onset untreated RA patients and three healthy controls (HCs). We then selected piRNAs related to immunoregulation by bioinformatics analysis and verified them in 42 new-onset RA patients and 81 HCs by RT-qPCR. Furthermore, a receiver operating characteristic curve was generated to quantify the diagnostic performance of these piRNAs. A correlation analysis was conducted to observe the link between piRNA expression and RA clinical characteristics. A total of 15 upregulated and 9 downregulated piRNAs among 1,565 known piRNAs were identified in peripheral leukocytes of RA patients. Dysregulated piRNAs were enriched in numerous pathways related to immunity. After selection and validation, two immunoregulation piRNAs (piR-hsa-27620 and piR-hsa-27124) were significantly elevated in RA patients and have good abilities to distinguish patients from controls, which have the potential to serve as biomarkers. PIWI and other proteins implicated in the piRNA pathway were also associated with RA.

Peripheral nervous system involvement in SARS-CoV-2 infection: a review of the current pediatric literature.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the pathogen responsible for the pandemic health emergency declared by the World Health Organization in March 2020. During the first part of the pandemic, adults showed mild to severe respiratory symptoms. Children seemed initially exempt, both from acute and subsequent complications. Hyposmia or anosmia were promptly identified as the main symptoms of acute infection, so neurotropism of SARS-CoV-2 was immediately suspected. (1, 2). As the emergency progressed, post infectious neurological complications were described also in pediatric population (3). Cases of cranial neuropathy in connection with acute SARS-CoV-2 infection have been reported in pediatric patients, as an isolate post infectious complication or in the context of the multisystem inflammatory syndrome in children (MIS-C) (4-6). Neuroinflammation is thought to be caused by several mechanisms, among which immune/autoimmune reactions (7), but so far, no specific autoantibody has been identified. SARS-CoV-2 can enter the central nervous system (CNS) directly and/or infect it retrogradely, through the peripheral nervous system (PNS), after replicating peripherally; several factors regulate invasion and subsequent neuroinflammation. Indeed, direct/secondary entry and replication can activate CNS-resident immune cells that, together with peripheral leukocytes, induce an immune response and promote neuroinflammation. In addition, as we will discuss in the following review, many cases of peripheral neuropathy (cranial and non-cranial) have been reported during or after SARS-CoV-2 infection. However, some authors have pointed out that the increase of cranial roots and ganglia in neurological imaging is not always observed in children with cranial neuropathy. (8). Even if a variety of case reports were published, opinions about an increased incidence of such neurologic diseases, linked to SARS-CoV-2 infection, are still controversial (9-11). Facial nerve palsy, ocular movements abnormalities and vestibular alterations are among the most reported issues in pediatric population (3-5). Moreover, an increased screen exposure imposed by social distancing led to acute oculomotion's disturbance in children, not primarily caused by neuritis (12, 13). The aim of this review is to suggest food for thought on the role of SARS-CoV-2 in neurological conditions, affecting the peripheral nervous system to optimize the management and care of pediatric patients.

Research Highlights.

Research Highlights.

Differential gene expression in peripheral leukocytes of pre-weaned Holstein heifer calves with respiratory disease.

Bovine respiratory disease (BRD) is a leading cause of calf morbidity and mortality, and prevalence remains high despite current management practices. Differential gene expression (DGE) provides detailed insight into individual immune responses and can illuminate enriched pathways and biomarkers that contribute to disease susceptibility and outcomes. The aims of this study were to investigate differences in peripheral leukocyte gene expression in Holstein preweaned heifer calves 1) with and without BRD, and 2) across weeks of age. Calves were enrolled for this short-term longitudinal study on two commercial dairies in Washington State. Calves were assessed every two weeks throughout the pre-weaning period using clinical respiratory scoring (CRS) and thoracic ultrasonography (TUS), and blood samples were collected. Calves were selected that were either healthy (n = 10) or had BRD diagnosed by CRS (n = 7), TUS (n = 6), or both (n = 6) in weeks 5 or 7 of life). Three consecutive time point samples were analyzed for each BRD calf consisting of PRE, ONSET, and POST samples. Nineteen genes of interest were selected based on previous gene expression studies in cattle: ALOX15, BPI, CATHL6, CXCL8, DHX58, GZMB, HPGD, IFNG, IL17D, IL1R2, ISG15, LCN2, LIF, MX1, OAS2, PGLYRP1, S100A8, SELP, and TNF. Comparisons were made between age and disease time point matched BRD and healthy calves as well as between calf weeks of age. No DGE was observed between diseased and healthy calves; however, DGE was observed between calf weeks of age regardless of disease state. Developmental differences in leukocyte gene expression, phenotype, and functionality make pre-weaned calves immunologically distinct from mature cattle, and early life shifts in calf leukocyte populations likely contribute to the age-related gene expression differences we observed. Age overshadows disease impacts to influence gene expression in young calves, and immune development progresses upon a common trajectory regardless of disease during the preweaning period.

Abstract 148: Lipoprotein Lipase Deficiency Reprograms Bone Marrow Cellular Metabolic Activity And Inflammatory Responses

Objectives: Activated bone marrow (BM) hematopoiesis is associated with systemic inflammation that increases risk for atherosclerosis. We had reported that lipoprotein lipase (LpL), in addition to being a lipolytic enzyme, regulated peripheral and tissue leukocyte levels and affected BM monocyte progenitor differentiation. LpL is mainly expressed in the specialized BM niches. We now questioned the role of LpL in influencing BM microenvironment that affects the homing and differentiation of hematopoietic stem/progenitor cells toward distinct lineages. Methods and Results: We characterized the metabolic and transcriptional phenotypes of BM cells utilizing immunofluorescence, flow cytometry, quantitative RT-PCR, and Seahorse Bioenergetics analysis in transgenic LpL knockout (KO) mice rescued by muscle-specific LpL expression. Differential gene expression (DEG) of BM cells were examined by bulk RNA sequencing (RNAseq). Our data indicate that LpL deficiency reduced expression of key BM vascular niche markers by 2-3-fold (p&lt;0.05), including CD31, Tie-2, endomucin, CXCL12, and CXCR4, as well their associated growth factors - HIF-1α and VEGF. BM vascular endothelial cadherin-labeled cells were decreased by &gt;50% (p&lt;0.05) in LpL KO when compared to that in WT. Cellular uptake of chylomicron-size lipid particles was reduced in LpL KO BM, which may link to metabolic adaptation from mitochondrial oxidative phosphorylation towards glycolysis observed in LpL KO BM (p&lt;0.05). Although the leptin-receptor positive perivascular cells were remained intact, expression of key lipolytic genes, such as ATGL, MGL, and LAL, were markedly reduced by &gt;50% in LpL KO (p &lt; 0.05). KEGG enrichment analysis of DEG shows that LpL KO BM cells had altered hematopoietic lineage signaling pathway. NFκB-directed signaling cascade was also downregulated in LpL KO BM cells, resulting in the reduced expression of pro-inflammatory TNF-α, IL-1β and IL-6. Conclusions: Our results indicate that LpL regulates metabolic activity and inflammatory responses of BM cells. A further understanding of these mechanisms will open the possibilities to target reprogram of both hematopoietic and BM niche cells as a therapeutic strategy to improve atherosclerosis-associated inflammation.

Differentially expressed miRNAs may help explaining the pathogenesis of relevant 22q11.2 clinical manifestations

Background and aimsChromosome 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion syndrome with a widely variable clinical spectrum, with >200 features reported. Genetic alone cannot explain the variability of the phenotype. DiGeorge Critical Region Gene 8 (DGCR8) encoded in the deleted region is crucial for miRNA biogenesis and therefore, for gene regulation. Aim of this study is to evaluate the role of miRNA in the clinical variability observed in 22q11.2DS. MethodsWe characterized miRNA expression profile in the peripheral leucocytes of 14 patients with a typical 3Mb deletion and 3 patients with a distal deletion not including TBX1 and DGCR8 compared to 10 age-matched healthy controls. Nine out of 17 patients had a familial form and came from 4 different families. ResultsThe unbiased hierarchical cluster analysis revealed significant clustering of the 22q11.2DS samples compared to normal controls. Samples from patients with distal deletion, clustered with patients with typical deletion. Twenty-nine differentially expressed miRNAs (padj< 0.05) were identified. As expected, the expression of miR185, encoded in the proximal part of the deleted region, was comparable to controls in 3 patients with distal deletion. High similarity of miRNA expression among patients coming from the same family was observed in 3 out of 4 families. Collectively, the 29 differentially expressed miRNA were predicted to regulate the expression of 13063 target genes, linked to multiple function and pathways. Interestingly, some DE-miRNAs are involved in the development of relevant 22q11.2 clinical manifestations, such as schizophrenia and autism (miR-132, miR-185-5p, miR-486-3p and miR-148-3p), B- and T-cell immunodysregulation and autoimmunity (miR-185-5p, miR-21-3p and miR-148-3p) and megakaryocytic differentiation (miR-486-5p). ConclusionsPatients with 22q11.2DS display a different miRNA expression profile compared to healthy controls. Some of the DE-miRNAs are involved in the development of relevant 22q11.2DS clinical manifestations, such as schizophrenia and autism, B- and T-cell immunodysregulation and autoimmunity and megakaryocytic differentiation and may be helpful to better define the pathogenesis of 22q11.2DS.