Paired Blood Samples Research Articles

Follow-up of humoral immune response after HPV vaccination using first-void urine: A longitudinal cohort study.

Assessment of humoral immune responses following human papillomavirus (HPV) vaccination currently relies on invasive blood sampling. This longitudinal cohort study explores the usability of first-void urine as a noninvasive alternative sample for antibody detection. In this study, 58 women receiving three doses of the 9vHPV vaccine within a Gardasil9 (9vHPV) Phase III randomized controlled trial were included. Participants provided paired first-void urine and blood samples before vaccination (M0), 1 month after the third dose (M7), and ~3 years after the third dose (M43). Type-specific antibody responses to the 9vHPV types were analyzed in 174 first-void urine and 172 serum samples using a virus-like particle-based IgG multiplex enzyme-linked immunosorbent assay. Additionally, total human IgG concentrations were determined using the BioPlex assay. At M7, 1 month after complete 9vHPV vaccination, 95%-100% of first-void urine and 100% of serum samples had detectable concentrations, varying by HPV type. At M43, 84%-100% of first-void urine and 98%-100% of serum samples had HPV-specific antibody concentrations. Results show significant Spearman rank correlations between type-specific HPV-antibody concentrations for paired first-void urine and serum at all time points. This study confirms the potential feasibility of utilizing first-void urine as a noninvasive immunological sample within HPV vaccine trials.

Exploration of Multiomic Profiles and Biomarkers as Predictors of Neoadjuvant Chemoradiotherapy Responsiveness in Esophageal Squamous Cell Carcinoma

The current gold standard of care for resectable locally advanced esophageal cancer is neoadjuvant chemoradiotherapy (NCRT) followed by surgery. Given that only 30-40% of patients with esophageal squamous cell carcinoma (ESCC) achieved a pathologic complete response (pCR) following neoadjuvant chemoradiotherapy, it is critical to understand the biological basis of NCRT resistance in esophageal cancer and identify biomarkers for these patients in order to further personalize treatment plans. We aim to depict the biological landscape of ESCC responsiveness and resistance to neoadjuvant chemoradiotherapy. Endoscopic biopsied specimens of the primary tumors and paired peripheral blood samples were obtained from 24 patients before neoadjuvant chemoradiotherapy and tested for whole exosome sequencing, RNA sequencing, and DIA mass spectrometry. Genomic data were analyzed for significantly mutated genes, copy number alterations, MSI, TMB, and mutational signatures. Transcriptomics and proteomics data were used to examine differentially activated pathways. GSEA and ActivePathways were used for the single omics level and joint multi-omics analysis, respectively. Tumor microenvironment (TME) characteristics were deconvoluted by xCell upon RNA-seq data. Treatment resistance biomarkers were identified and validated in a separate cohort using mIHC. In the study cohort, 54% (13/24) of the patients achieved pCR. WES data suggested that FBXW7 was more frequently mutated in the pCR group (fisher test p-value = 0.029), and the most significant cytoband loss in the pCR group was 9p21.3 (qval = 0.001). Differences in TMB, MSI, and mutational signatures were not significant between groups. Combined transcriptomics and proteomics analysis revealed that type I interferon signaling pathways and RIG-I-like receptor signaling pathways(p<0.05) were enriched in non-pCR tumors. Esophageal cancer cohort RNA-seq data from TCGA verified the correlation between the genetic variances (FBXW7 mutation and 9p21.3 loss) and the decreased expression of type I interferon signaling pathway genes. In TME analysis, tolerogenic dendritic cells and exhausted T cell signatures were significantly enriched in non-pCR tumors, indicating an immunosuppressive status in treatment resistant patients. Based on proteomics PPI network and differential expression genes from RNA-seq data, a biomarker panel consisted of 12 proteins predictive of non-pCR tumors was identified: STAT1, EIF2AK2, MX1, BST2, TRIM21, SAMHD1, IFI44L, GBP1, PARP14, ISG15, IFIT3, and HLA-B. The expression of selected genes was validated by mIHC in an independent cohort. Through a multiomics approach, we described the biological characteristics of ESCC with distinct responses to neoadjuvant chemoradiotherapy and proposed a panel of 12 proteins as predictive biomarkers for non-pCR patients.

Induced Abortion and the Risk of Rh Sensitization

While population-level data suggest Rh immunoglobulin is unnecessary before 12 weeks' gestation, clinical evidence is limited. Thus, guidelines vary, creating confusion surrounding risks and benefits of Rh testing and treatment. As abortion care in traditional clinical settings becomes harder to access, many people are choosing to self-manage and need to know if ancillary blood type testing is necessary. To determine how frequently maternal exposure to fetal red blood cells (fRBCs) exceeds the most conservative published threshold for Rh sensitization in induced first-trimester abortion. Multicenter, observational, prospective cohort study using high-throughput flow cytometry to detect circulating fRBCs in paired maternal blood samples before and after induced first-trimester abortion (medication or procedural). Individuals undergoing induced first-trimester abortion before 12 weeks 0 days' gestation were included. Paired blood samples were available from 506 participants who underwent either medical (n = 319 [63.0%]) or procedural (n = 187 [37.0%]) abortion. Induced first-trimester abortion. The primary outcome was the proportion of participants with fRBC counts above the sensitization threshold (125 fRBCs/5 million total RBCs) after induced first-trimester abortion. Among the 506 participants, the mean (SD) age was 27.4 (5.5) years, 313 (61.9%) were Black, and 123 (24.3%) were White. Three of the 506 participants had elevated fRBC counts at baseline; 1 of these patients had an elevated fRBC count following the abortion (0.2% [95% CI, 0%-0.93%]). No other participants had elevated fRBC counts above the sensitization threshold after induced first-trimester abortion. The median change from baseline was 0 fRBCs, with upper 95th and 99th percentiles of 24 and 35.6 fRBCs, respectively. Although there was a strong association between the preabortion and postabortion fRBC counts, no other baseline characteristic was significantly associated with postabortion fRBC count. Induced first-trimester abortion is not a risk factor for Rh sensitization, indicating that Rh testing and treatment are unnecessary before 12 weeks' gestation. This evidence may be used to inform international guidelines for Rh immunoglobulin administration following first-trimester induced abortion.

Transcriptome from Paired Samples Improves the Power of Comprehensive COVID-19 Host-Viral Characterization.

Previous transcriptome profiling studies showed significantly upregulated genes and altered biological pathways in acute COVID-19. However, changes in the transcriptional signatures during a defined time frame are not yet examined and described. The aims of this study included viral metagenomics and evaluation of the total expression in time-matched and tissue-matched paired COVID-19 samples with the analysis of the host splicing profile to reveal potential therapeutic targets. Prospective analysis of paired nasopharyngeal swabs (NPS) and blood (BL) samples from 18 COVID-19 patients with acute and resolved infection performed using Kallisto, Suppa2, Centrifuge, EdgeR, PantherDB, and L1000CDS2 tools. In NPS, we discovered 6 genes with changed splicing and 40 differentially expressed genes (DEG) that yielded 88 altered pathways. Blood samples yielded 15 alternatively spliced genes. Although the unpaired DEG analysis failed, pairing identified 78 genes and 242 altered pathways with meaningful clinical interpretation and new candidate drug combinations with up to 65% overlap. Metagenomics analyses showed SARS-CoV-2 dominance during and after the acute infection, with a significant reduction in NPS (0.008 vs. 0.002, p = 0.019). Even though both NPS and BL give meaningful insights into expression changes, this is the first demonstration of how the power of blood analysis is vastly maximized by pairing. The obtained results essentially showed that pairing is a determinant between a failed and a comprehensive study. Finally, the bioinformatics results prove to be a comprehensive tool for full-action insights, drug development, and infectious disease research when designed properly.

Combined Molecular and Morphological Imaging of CTCs for HER2-Targeted Chemotherapy Guidance.

Since biomolecules change dynamically with tumor evolution and drug treatment, it is necessary to confirm target molecule expression in real time for effective guidance of subsequent chemotherapy treatment. However, current methods to confirm target proteins require complex processing steps and invasive tissue biopsies, limiting their clinical utility for targeted treatment monitoring. Here, CTCs, as a promising liquid biopsy source, were used to molecularly characterize the target protein HER2. To accurately identify CTCs, we specifically proposed a combined molecular and morphological imaging method, rather than using specific biomarker alone or morphology analysis, we identified CTCs as CK19+/CD45-/HE+. On the basis of the accurate identification of CTCs, we further analyzed the target protein HER2 in clinical patients at the single-CTC level. Comparative analysis of the clinical results of patient pathological tissue and paired blood samples showed that CTCs had a heterogeneous HER2 expression at the single-cell level and showed results inconsistent with the immunohistochemistry results in some cases. CTC-based analysis could help clinicians have a more comprehensive understanding of patient target protein expression. We believe that CTC-based target protein studies are of great significance for the precise management of targeted therapy.

A bicentric cohort study comparing umbilical cord and neonatal blood samples for chemistry tests at birth.

To compare umbilical cord and neonatal blood for chemistry tests upon admission to the neonatal intensive care unit (NICU). We designed a prospective, bicentric cohort study enrolling newborns (n = 71) with a planned admission to the NICU. Paired samples of umbilical cord and infant's blood were collected, analyzed, and compared. An intraclass correlation coefficient (ICC) was calculated for a repeatability analysis, and a Bland-Altman analysis was performed to assess the agreement between the 2 methods of sampling. The multivariable coefficient of determination (R2) was reported to quantify the degree of correlation between the methods of measurement. The degree of agreement between the 2 sampling methods for chemistry tests was fair to good for high-sensitivity C-reactive protein (ICC = 0.79 [95% CI, 0.67-0.87]), phosphorus (ICC = 0.83 [95% CI, 0.73-0.90]), and albumin (ICC = 0.76 [95% CI, 0.60-0.86]), while it was good to excellent for γ-glutamyl transpeptidase (ICC = 0.95 [95% CI, 0.88-0.98]) and procalcitonin (ICC = 0.90 [95% CI, 0.76-0.96]). Umbilical cord blood is a reliable replacement source for multiple chemistry tests at birth. This sampling method has the potential to minimize the risk of transfusion-requiring anemia in newborns and its associated complications. Further studies are warranted to assess the efficacy of this strategy in improving neonatal outcomes.

A single-cell transcriptional landscape of immune cells shows disease-specific changes of T cell and macrophage populations in human achalasia

Achalasia is a rare motility disorder of the esophagus caused by the gradual degeneration of myenteric neurons. Immune-mediated ganglionitis has been proposed to underlie the loss of myenteric neurons. Here, we measure the immune cell transcriptional profile of paired lower esophageal sphincter (LES) tissue and blood samples in achalasia and controls using single-cell RNA sequencing (scRNA-seq). In achalasia, we identify a pattern of expanded immune cells and a specific transcriptional phenotype, especially in LES tissue. We show C1QC+ macrophages and tissue-resident memory T cells (TRM), especially ZNF683+ CD8+ TRM and XCL1+ CD4+ TRM, are significantly expanded and localized surrounding the myenteric plexus in the LES tissue of achalasia. C1QC+ macrophages are transcriptionally similar to microglia of the central nervous system and have a neurodegenerative dysfunctional phenotype in achalasia. TRM also expresses transcripts of dysregulated immune responses in achalasia. Moreover, inflammation increases with disease progression since immune cells are more activated in type I compared with type II achalasia. Thus, we profile the immune cell transcriptional landscape and identify C1QC+ macrophages and TRM as disease-associated immune cell subsets in achalasia.

Identification of intratumoral and peripheral T-cell receptor (TCR) repertoire features associated with acquired (Ar) and primary (Pr) resistance to immune checkpoint inhibitors (ICI).

59 Background: Our understanding of the mechanisms of Pr vs Ar to ICI is limited. T-cells are the main driver of ICI response. Therefore, interrogation of intratumoral and peripheral TCR repertoire could expand our comprehension of T-cell mediated factors underlying Pr and Ar to ICI. Methods: The Immune Resistance Interrogation Study (NCT04243720) is a prospective study to comprehensively characterize cancers with Pr vs Ar to ICI (Genta et. al., ASCO 2021). We conducted TCRβ capture and sequencing (CapTCR-seq) on paired tumors and blood samples collected from solid tumor patients (pts) at the time of progression on ICI. Tumor and peripheral TCR diversities were calculated for each pt. Grouping Lymphocyte Interactions by Paratope Hotspots (GLIPHII) was used to investigate the potential antigen specificities of intratumoral T-cells. Pt-derived TCRs were pooled with TCRs from a public database with known antigen specificities (TCRdb). GLIPHII grouped pt-derived and external TCRs with similar CDR3s (hypervariable TCR domains) in clusters. The clusters were then converted into a network model representing unique TCRs as nodes. The number of connections between each pt-derived TCR and similar TCRs from TCRdb was defined as node degree. Comparisons between groups were done using Mann Whitney U Test. Results: As of February 2023, 75 pts (45 Pr/30 Ar) were enrolled. CapTCR-Seq in blood and tumor samples was completed in 13 pts (7Pr/6Ar) with the following characteristics: median age 57 years (26-77), 8 male (62%), 8 melanoma (62%), 4 HNSCC (31%), and 1 GE-junction (7%). 5 pts received PD-1/PD-L1 inhibitor monotherapy (38%), 8 pts ICl-based combinations (62%). No significant differences in intratumoral (10.3 [1.3-34.2] vs 15.2 [6.6-32.4] p=0.29) and peripheral (145.5 [65.3- 521.9] vs 136.0 [35.1-474.4] p=0.53) Shannon diversities were observed between Pr and Ar. Twelve of 13 (92%) pts had at least 1 intratumoral TCR clustered with TCRdb derived TCRs. A higher median of node degree was observed in non-melanoma tumors (5.5 [2-12] vs 2.0 [1-4] p=0.03). A trend towards a higher median of node degree was reported in Ar vs Pr (5.5 [1-12] vs 2.0 [1-4] p=0 .07 ). Some of the GLIPHll-identified clusters contained TCRs derived from multiple Ar pts. Conclusions: lntratumoral TCRs from non-melanoma and Ar pts had a higher node degree, indicating similarity with a larger number of external TCRs. The difference between Pr and Ar was not significant, potentially due to the small sample size. Similar TCRs belonging to the same GLIPHll-identified clusters were shared among multiple Ar pts. If confirmed in a larger dataset, these findings might suggest the existence of a set of intratumoral exhausted T-cells shared by multiple pts contributing to Ar but not to Pr. Pt accrual, sample collection and analysis are ongoing. Additional data will be presented.

The complementarity of phosphatidylethanol in whole blood and ethyl glucuronide in hair as biomarkers for the monitoring of alcohol use.

Monitoring long-term alcohol use and/or abstinence is essential in clinical and medico-legal cases. Analysis of ethyl glucuronide (EtG) in hair provides information on alcohol consumption over several months. However, there is a lag time between ethanol consumption, incorporation of EtG in the hair bulb and hair growing out of the scalp. Phosphatidylethanol (PEth) 16:0/18:1 analysis in whole blood has a detection window of 2-4 weeks, allowing for the detection of recent alcohol consumption. In this study, 2340 paired samples (of hair and venous whole blood from 1170 individuals) were analysed for EtG in hair (hEtG) and PEth 16:0/18:1 in venous whole blood. PEth 16:0/18:1 and hEtG results were subdivided into three categories according to the consensus of SoHT (hEtG) and PEth-NET (PEth): abstinence/low, moderate or excessive alcohol consumption. For hEtG analysis, 446 individuals presented abstinence/low alcohol consumption, of which 2% were classified as excessive alcohol users through PEth 16:0/18:1 analysis. This suggests excessive alcohol consumption in the weeks before sample collection. Out of 483 individuals classified as heavy alcohol users based on hEtG analysis, 14% showed abstinence/low alcohol consumption for PEth 16:0/18:1 analysis, implying that these subjects stopped drinking 2-4 weeks before sample collection. Our results show that the analysis of the two different biomarkers can lead to a more accurate categorisation of individuals. Therefore, we emphasize that for the retrospective investigation of alcohol use, it is necessary to include two alcohol use biomarkers with different detection windows.

Are high avidity antibodies to Plasmodium falciparum antigens preferentially transferred across the placenta of premature and term babies?

IntroductionTransplacental transport of maternal IgG via the neonatal Fc receptor (FcRn) provides babies with passive immunity. Several factors are reported to influence transport, including the avidity of antibodies (Abs) for their cognate antigens. Unfortunately, information on the role of antibody (Ab) avidity is limited. This study investigated if i) antibodies (Abs) with high avidity for 6 Plasmodium falciparum antigens and tetanus toxoid (TTx) were preferentially transferred to premature and term Cameroonian babies and ii) if Ab avidity was increased in babies whose mothers had placental malaria (PM), implicating the involvement of immune complexes. MethodsTotal IgG (mg/ml) and Abs to malarial antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3, DBL5 of VAR2CSA) and TTx were measured in paired mother-cord samples obtained from premature and term deliveries in Cameroon. Half the women had PM at delivery. Avidity Indices (AIs) were determined by treating antigen-bound-Abs with different molar concentrations of NH4SCN and calculating 50% endpoints. ResultsTotal IgG and antigen-specific Abs increased in cord blood with gestational age; however, AIs did not. AIs in paired maternal-cord blood samples were strongly associated for all antigens (r = 0.77–0.96). However, no significant different in AIs was found between paired mother-cord blood samples for any of the antigens (p values > 0.05). Similarly, Ab avidity was not increased in cord blood of babies whose mothers had PM or hypergammaglobulinemia. DiscussionOverall, there was no evidence that higher avidity Abs to any of the malarial antigens or TTx were preferentially transferred to Cameroonian babies.

Changes in methylation-based aging in women who do and do not develop breast cancer.

Breast cancer survivors have increased incidence of age-related diseases, suggesting that some survivors may experience faster biological aging. Among 417 women enrolled in the prospective Sister Study cohort, DNA methylation data were generated on paired blood samples collected an average of 7.7 years apart and used to calculate 3 epigenetic metrics of biological aging (PhenoAgeAccel, GrimAgeAccel, and Dunedin Pace of Aging Calculated from the Epigenome [DunedinPACE]). Approximately half (n = 190) the women sampled were diagnosed and treated for breast cancer between blood draws, whereas the other half (n = 227) remained breast cancer-free. Breast tumor characteristics and treatment information were abstracted from medical records. Among women who developed breast cancer, diagnoses occurred an average of 3.5 years after the initial blood draw and 4 years before the second draw. After accounting for covariates and biological aging metrics measured at baseline, women diagnosed and treated for breast cancer had higher biological aging at the second blood draw than women who remained cancer-free as measured by PhenoAgeAccel (standardized mean difference [β] = 0.13, 95% confidence interval [CI) = 0.00 to 0.26), GrimAgeAccel (β = 0.14, 95% CI = 0.03 to 0.25), and DunedinPACE (β = 0.37, 95% CI = 0.24 to 0.50). In case-only analyses assessing associations with different breast cancer therapies, radiation had strong positive associations with biological aging (PhenoAgeAccel: β = 0.39, 95% CI = 0.19 to 0.59; GrimAgeAccel: β = 0.29, 95% CI = 0.10 to 0.47; DunedinPACE: β = 0.25, 95% CI = 0.02 to 0.48). Biological aging is accelerated following a breast cancer diagnosis and treatment. Breast cancer treatment modalities appear to differentially contribute to biological aging.

Cytotoxic T cell subsets in peripheral blood and cerebrospinal fluid from patients with multiple sclerosis

Using multicolor flow cytometry, the main cytotoxic T lymphocytes (Tcyt) subsets were identified, based on the expression of CD45RA and CD62L in paired samples of peripheral blood and cerebrospinal fluid from the patients during the relapse (n = 32) and remission (n = 20) of multiple sclerosis (MS), as well as in the peripheral blood samples of healthy volunteers (n = 51). During the relapse of MS, we have observed a decreased relative number of CD3+CD4+ cells and CD4/CD8 ratio in cerebrospinal liquor. In peripheral blood taken from the relapsed MS patients, we have found significant correlations between EDSS score and absolute counts (r = -0,430, p = 0.014), and with relative numbers of CD45RA+CD62L+Tcyt (r = -0,502, p = 0.003). In remission state of MS, the relative numbers of blood CD45RA-CD62L-Tcyt cells exhibited a significant decrease (p = 0.005) to 8.70% (6.51-11.63) against control group with 12.18% (10.38-15.24), although it did not significantly differ (p = 0.114) from the relapsed patients with 11.31% (8.28-13.90). Studies of liquor samples have shown that, during MS relapse, the percentage of CD45RA-CD62L-Tcyt was increased (p = 0.027) up to 8.16% (6.40-11.40), while in remission state these cells comprised only 6.49% (4.51-8.39) from the total CD3+ cell number. During relapse of MS, some positive correlations were revealed between the relative number of nave, CM, EM and TEMRA Tcyt from liquor, and the percentages, as well as contents of similar T cell subsets in peripheral blood samples. The inverse relationship between the level of EM Tcyt from liquor and peripheral blood naive cells showed the close relationship between these two Tcyt subsets and clinical manifestations of MS (i.e., scores of EDSS scale). During the remission period, most of these correlations are disrupted. Further investigations of cytotoxic T cells dynamics in peripheral blood and cerebrospinal fluid will help to approach the understanding of MS pathogenesis by revealing novel markers for the clinical prognosis in this disorder.

Clinical significance of determining the hypermethylation of the RUNX3 gene promoter and its cohypermethylation with the BRCA1 gene for patients with breast cancer.

The aim of this study was to assess the clinical significance of RUNX3 gene hypermethylation in the pathogenetic mechanisms of breast cancer in women, taking into account its cohypermethylation with the BRCA1 gene. This study included 74 women with newly diagnosed breast cancer (samples from female primary breast carcinomas and paired peripheral blood samples) and 62 women without oncological pathology-control group (peripheral blood samples). Epigenetic testing for hypermethylation status studying was performed in all samples on freshly collected material with the addition of a preservative before the storage and DNA isolation. Hypermethylation of the RUNX3 gene promoter region was detected in 71.6% samples of breast cancer tissue and in 35.13% samples of blood. The RUNX3 gene promoter region hypermethylation was significantly higher among breast cancer patients compared to the control group. The frequency of cohypermethylation in RUNX3 and BRCA1 genes was significantly increased in breast cancer tissues compared to the blood of patients. A significantly increased frequency of the hypermethylation of the RUNX3 gene promoter region and its cohypermethylation with the BRCA1 gene promoter region was found in tumor tissue and blood samples from patients with breast cancer, in contrast to the control group. The identified differences indicate the importance of further investigations of suppressor genes cohypermethylation in patients with breast cancer. Further large-scale studies are needed to find out whether the detected hypermethylation and cohypermethylation of the RUNX3 gene promoter region will have an impact on the treatment strategy in patients.

AXL+SIGLEC6+ dendritic cells in cerebrospinal fluid and brain tissues of patients with autoimmune inflammatory demyelinating disease of CNS

Inflammatory demyelinating disease of the CNS (IDD) is a heterogeneous group of autoimmune diseases, and multiple sclerosis is the most common type. Dendritic cells (DCs), major antigen-presenting cells, have been proposed to play a central role in the pathogenesis of IDD. The AXL+SIGLEC6+ DC (ASDC) has been only recently identified in humans and has a high capability of T cell activation. Nevertheless, its contribution to CNS autoimmunity remains still obscure. Here, we aimed to identify the ASDC in diverse sample types from IDD patients and experimental autoimmune encephalomyelitis (EAE). A detailed analysis of DC subpopulations using single-cell transcriptomics for the paired cerebrospinal fluid (CSF) and blood samples of IDD patients (total n = 9) revealed that three subtypes of DCs (ASDCs, ACY3+ DCs, and LAMP3+ DCs) were overrepresented in CSF compared with their paired blood. Among these DCs, ASDCs were also more abundant in CSF of IDD patients than in controls, manifesting poly-adhesional and stimulatory characteristics. In the brain biopsied tissues of IDD patients, obtained at the acute attack of disease, ASDC were also frequently found in close contact with T cells. Lastly, the frequency of ASDC was found to be temporally more abundant in acute attack of disease both in CSF samples of IDD patients and in tissues of EAE, an animal model for CNS autoimmunity. Our analysis suggests that the ASDC might be involved in the pathogenesis of CNS autoimmunity.

Single-cell clonal tracing of glandular and circulating T cells identifies a population of CD9+ CD8+ T cells in primary Sjogren's syndrome.

Primary Sjogren's syndrome (pSS) is a complex chronic autoimmune disease in which local tissue damage in exocrine glands is combined with broader systemic involvement across the body in tissues including the skin. These combined manifestations negatively impact patient health and quality of life. While studies have previously reported differences in immune cell composition in the peripheral blood of pSS patients relative to healthy control subjects, a detailed immune cell landscape of the damaged exocrine glands of these patients remains lacking. Through single-cell transcriptomics and repertoire sequencing of immune cells in paired peripheral blood samples and salivary gland biopsies, we present here a preliminary picture of adaptive immune response in pSS. We characterize a number of points of divergence between circulating and glandular immune responses that have been hitherto underappreciated, and identify a novel population of CD8+ CD9+ cells with tissue-residential properties that are highly enriched in the salivary glands of pSS patients. Through comparative analyses with other sequencing data, we also observe a potential connection between these cells and the tissue-resident memory cells found in cutaneous vasculitis lesions. Together, these results indicate a potential role for CD8+ CD9+ cells in mediating glandular and systemic effects associated with pSS and other autoimmune disorders.

Overdiagnosis of Newborn Hyperbilirubinemia: A Natural Experiment in Quality Improvement Fundamentals.

In January 2020, 2 hospitals (A and B) transitioned to a new method of measuring TSB using a new clinical chemistry analyzer (Siemens Atellica CH), which measured TSB by vanadate oxidase assay instead of the previous diazo assay. Five affiliated hospitals (C-G) continued to utilize the diazo assay. This natural experiment led to a comparison of data across the 7 hospitals. We analyzed: (1) TSB levels, (2) hospital hyperbilirubinemia readmissions, and (3) paired TSB measurements comparing the diazo assay and vanadate oxidase method. Compared to the 2019 baseline, Hospitals A and B had a significant increase in TSBs ≥17.0 mg/dl and TSBs ≥20 mg/dl in 2020; Hospitals C-G did not. Readmissions for phototherapy significantly increased in hospitals A and B in 2020 compared to 2019. Paired blood samples showed bias-elevated TSBs by vanadate assay compared to the diazo method. By 2021, the laboratory resumed processing TSB samples by diazo assay, and the frequency of elevated TSBs and hyperbilirubinemia readmissions returned to 2019 levels. Factitious TSB elevation related to an assay change significantly increased newborn hyperbilirubinemia evaluations and phototherapy readmissions. Imbedded quality improvement methodologies of careful structure, process, and outcomes review hastened resolution.

PFAS levels in paired drinking water and serum samples collected from an exposed community in Central North Carolina

The community of Pittsboro, North Carolina has been documented to have extensive per- and polyfluoroalkyl substances (PFAS) contamination in its drinking water source, the Haw River, over the last 20 years. However, a detailed exposure assessment has never been conducted. In this study, we sought to characterize the PFAS in paired drinking water and blood samples collected from a small cohort of Pittsboro residents (n = 49). Drinking water and serum from blood were collected from adults in late 2019 and early 2020 and were analyzed to quantify 13 PFAS analytes. In order to explore potential health effects of PFAS exposure, serum was further analyzed for clinical chemistry endpoints that could be potentially associated with PFAS (e.g., cholesterol, liver function biomarkers). PFAS were detected in all serum samples, and some serum PFAS concentrations were 2 to 4 times higher than the median U.S. serum concentrations reported in the general U.S. population. Of the 13 PFAS in drinking water, perfluorohexanoic acid (PFHxA) was measured at the highest concentrations. PFAS levels in the current drinking water were not associated with current serum PFAS, suggesting that the serum PFAS in this cohort likely reflects historical exposure to PFAS with long half-lives (e.g., PFOS and PFOA). However, one PFAS with a shorter half-life (PFHxA) was observed to increase in serum, reflecting the temporal variability of PFHxA in river and drinking water. Statistical analyses indicated that serum PFOA and PFHxS were positively associated with total cholesterol and non-HDL cholesterol. No serum PFAS was associated with HDL cholesterol. In the clinical chemistry analyses, serum PFHxA was found to be negatively associated with electrolytes and liver enzymes (e.g., AST and ALT), and serum PFOS was found to be positively associated with the ratio of blood urea nitrogen to creatinine (BUN:Cre). While small in size, this study revealed extensive exposure to PFAS in Pittsboro and associations with clinical blood markers, suggesting potential health impacts in community residents.

Increased PDGFRB and NF-κB signaling caused by highly prevalent somatic mutations in intracranial aneurysms.

Intracranial aneurysms (IAs) are a high-risk factor for life-threatening subarachnoid hemorrhage. Their etiology, however, remains mostly unknown at present. We conducted screening for sporadic somatic mutations in 65 IA tissues (54 saccular and 11 fusiform aneurysms) and paired blood samples by whole-exome and targeted deep sequencing. We identified sporadic mutations in multiple signaling genes and examined their impact on downstream signaling pathways and gene expression in vitro and an arterial dilatation model in mice in vivo. We identified 16 genes that were mutated in at least one IA case and found that these mutations were highly prevalent (92%: 60 of 65 IAs) among all IA cases examined. In particular, mutations in six genes (PDGFRB, AHNAK, OBSCN, RBM10, CACNA1E, and OR5P3), many of which are linked to NF-κB signaling, were found in both fusiform and saccular IAs at a high prevalence (43% of all IA cases examined). We found that mutant PDGFRBs constitutively activated ERK and NF-κB signaling, enhanced cell motility, and induced inflammation-related gene expression in vitro. Spatial transcriptomics also detected similar changes in vessels from patients with IA. Furthermore, virus-mediated overexpression of a mutant PDGFRB induced a fusiform-like dilatation of the basilar artery in mice, which was blocked by systemic administration of the tyrosine kinase inhibitor sunitinib. Collectively, this study reveals a high prevalence of somatic mutations in NF-κB signaling pathway-related genes in both fusiform and saccular IAs and opens a new avenue of research for developing pharmacological interventions.

CBPH assay for the highest sensitive detection of SARS-COV-2 in the semen

BackgroundGreat concerns have been raised on SARS-CoV-2 impact on men's andrological well-being, and many studies have attempted to determine whether SARS-CoV-2 is present in the semen and till now the data are unclear and somehow ambiguous. However, these studies used quantitative real-time (qRT) PCR, which is not sufficiently sensitive to detect nucleic acids in clinical samples with a low viral load. MethodsThe clinical performance of various nucleic acid detection methods (qRT-PCR, OSN-qRT-PCR, cd-PCR, and CBPH) was assessed for SARS-CoV-2 using 236 clinical samples from laboratory-confirmed COVID-19 cases. Then, the presence of SARS-CoV-2 in the semen of 12 recovering patients was investigated using qRT-PCR, OSN-qRT-PCR, cd-PCR, and CBPH in parallel using 24 paired semen, blood, throat swab, and urine samples. ResultsThe sensitivity and specificity along with AUC of CBPH was markedly higher than the other 3methods. Although qRT-PCR, OSN-qRT-PCR and cdPCR detected no SARS-CoV-2 RNA in throat swab, blood, urine, and semen samples of the 12 patients, CBPH detected the presence of SARS-CoV-2 genome fragments in semen samples, but not in paired urine samples, of 3 of 12 patients. The existing SARS-CoV-2 genome fragments were metabolized over time. ConclusionsBoth OSN-qRT-PCR and cdPCR had better performance than qRT-PCR, and CBPH had the highest diagnostic performance in detecting SARS-CoV-2, which contributed the most improvement to the determination of the critical value in gray area samples with low vrial load, which then provides a rational screening strategy for studying the clearance of coronavirus in the semen over time in patients recovering from COVID-19. Although the presence of SARS-CoV-2 fragments in the semen was demonstrated by CBPH, COVID-19 is unlikely to be sexually transmitted from male partners for at least 3 months after hospital discharge.

The role of innate immune system in modulating CHK1 inhibitor (CHK1i) response in BRCA wild-type (BRCAwt), platinum-resistant high-grade serous ovarian cancer (PR-HGSOC): Exploratory analysis from a phase II study of CHK1i prexasertib.

5557 Background: Immune checkpoint blockade, e.g., anti-PD-1/PD-L1, demonstrated limited efficacy in HGSOC despite high levels of tumor-infiltrating lymphocytes, suggesting the innate, rather than adaptive, immune system may play a prominent role in anticancer response. Preclinical studies suggest CHK1i modulates the innate immune milieu. We previously showed clinical activity of prexasertib in BRCAwt, PR-HGSOC patients (pts). Here, we investigated the association of innate immunogenic features with CHK1i response. Methods: Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. Baseline (BL) fresh core biopsies were collected from pts enrolled in the biopsy (Bi) cohort for RNA sequencing. Computational immunogenomic analysis (CIBERSORT) was used for immune cell fractions quantification. Blood samples (BS) were obtained at BL and cycle 1 day 15 (C1D15) from pts enrolled in both Bi and non-Bi (nBi) cohorts for multiparametric flow cytometry (FC) analysis to evaluate dynamic changes in immune cell subsets. Mann-Whitney and Wilcoxon tests were used to compare unpaired and paired data, respectively. Results: 49 BRCAwt, PR-HGSOC pts were enrolled in the Bi cohort (n=25) and nBi cohort (n=24). 39 were evaluable per RECIST v1.1. 18/39 pts (46.2%) had a clinical benefit (CB), defined as progression-free survival≥6 months. Transcriptomic profiles of BL biopsies (n=15) met the quality control and assurance (Gene Similarity Index, Principal Component Analysis) for further in silico analysis. CIBERSORT analysis did not exhibit any differences in the immune cell fractions between CB and non-CB (NCB) groups at BL. FC analysis on paired BS (BL and C1D15) showed an increase of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs, p&lt;0.001) and classical monocytes (CM, p=0.003) in the NCB group (18 paired BS). In the CB group (19 paired BS), CD141+ and CD1c+ dendritic cells (DCs) express higher rates of CD83, a marker of maturity and functionality (p&lt;0.001 and p&lt;0.001, respectively), despite no increase in the absolute number of DCs. At C1D15, a higher rate of M-MDSCs (median 7.8% vs. 5.52%; p=0.03) and CM (median 40.7% vs. 31.7%; p=0.03) were found in the NCB group (n=18) compared to the CB group (n=19); also, CM showed lower expression of HLA-DR in the NCB group (median 19.25% vs. 27.8%; p=0.02). Conclusions: Collectively, our data suggest the possible involvement of innate immunity in modulating CHK1i response. Increasing peripheral immunosuppressive cells and lower immunocompetence of the innate immune system may contribute to CHK1i resistance, whereas the functionality of DCs may be involved in the response. Enhancing innate immunity may represent a strategy to increase CHK1i response in this hard-to-treat population. Clinical trial information: NCT02203513 .