Quantification of DNA in Neonatal Dried Blood Spots by Adenine Tandem Mass Spectrometry.

Newborn screening programs have expanded to include molecular-based assays as first-tier tests and the success of these assays depends on the quality and yield of DNA extracted from neonatal dried blood spots (DBS). To meet high throughput and rapid turnaround time requirements, newborn screening laboratories adopted rapid DNA extraction methods that produce crude extracts. Quantification of DNA in neonatal DBS is not routinely performed due to technical challenges; however, this may enhance the performance of assays that are sensitive to amounts of input DNA. In this study, we developed a novel high throughput method to quantify total DNA in DBS. It is based on specific acid-catalyzed depurination of DNA followed by mass spectrometric quantification of adenine. The amount of adenine was used to calculate DNA quantity per 3.2 mm DBS. Reference intervals were established using archived, neonatal DBS (n = 501) and a median of 130.6 ng of DNA per DBS was obtained, which is in agreement with literature values. The intra- and interday variations were <15%. The limits of detection and quantification were 12.5 and 37.8 nmol/L adenine, respectively. We demonstrated that DNA from neonatal DBS can be successfully quantified in high throughput settings using instruments currently deployed in NBS laboratories.

High SARS-CoV-2 seroincidence but low excess COVID mortality in Sierra Leone in 2020-2022.

While SARS-CoV-2 infection appears to have spread widely throughout Africa, documentation of associated mortality is limited. We implemented a representative serosurvey in one city of Sierra Leone in Western Africa, paired with nationally representative mortality and selected death registration data. Cumulative seroincidence using high quality SARS-CoV-2 serological assays was 69% by July 2021, rising to 84% by April 2022, mostly preceding SARS-CoV-2 vaccination. About half of infections showed evidence of neutralizing antibodies. However, excess death rates were low, and were concentrated at older ages. During the peak weeks of viral activity, excess mortality rates were 22% for individuals aged 30-69 years and 70% for those over 70. Based on electronic verbal autopsy with dual independent physician assignment of causes, excess deaths during viral peaks from respiratory infections were notable. Excess deaths differed little across specific causes that, a priori, are associated with COVID, and the pattern was consistent among adults with or without chronic disease risk factors. The overall 6% excess of deaths at ages ≥30 from 2020-2022 in Sierra Leone is markedly lower than reported from South Africa, India, and Latin America. Thus, while SARS-CoV-2 infection was widespread, our study highlights as yet unidentified mechanisms of heterogeneity in susceptibility to severe disease in parts of Africa.

Hybrid immunity from severe acute respiratory syndrome coronavirus 2 infection and vaccination in Canadian adults: A cohort study.

Background: Few national-level studies have evaluated the impact of 'hybrid' immunity (vaccination coupled with recovery from infection) from the Omicron variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: From May 2020 to December 2022, we conducted serial assessments (each of ~4000-9000 adults) examining SARS-CoV-2 antibodies within a mostly representative Canadian cohort drawn from a national online polling platform. Adults, most of whom were vaccinated, reported viral test-confirmed infections and mailed self-collected dried blood spots (DBSs) to a central lab. Samples underwent highly sensitive and specific antibody assays to spike and nucleocapsid protein antigens, the latter triggered only by infection. We estimated cumulative SARS-CoV-2 incidence prior to the Omicron period and during the BA.1/1.1 and BA.2/5 waves. We assessed changes in antibody levels and in age-specific active immunity levels. Results: Spike levels were higher in infected than in uninfected adults, regardless of vaccination doses. Among adults vaccinated at least thrice and infected more than 6 months earlier, spike levels fell notably and continuously for the 9-month post-vaccination. In contrast, among adults infected within 6 months, spike levels declined gradually. Declines were similar by sex, age group, and ethnicity. Recent vaccination attenuated declines in spike levels from older infections. In a convenience sample, spike antibody and cellular responses were correlated. Near the end of 2022, about 35% of adults above age 60 had their last vaccine dose more than 6 months ago, and about 25% remained uninfected. The cumulative incidence of SARS-CoV-2 infection rose from 13% (95% confidence interval 11-14%) before omicron to 78% (76-80%) by December 2022, equating to 25 million infected adults cumulatively. However, the coronavirus disease 2019 (COVID-19) weekly death rate during the BA.2/5 waves was less than half of that during the BA.1/1.1 wave, implying a protective role for hybrid immunity. Conclusions: Strategies to maintain population-level hybrid immunity require up-to-date vaccination coverage, including among those recovering from infection. Population-based, self-collected DBSs are a practicable biological surveillance platform. Funding: Funding was provided by the COVID-19 Immunity Task Force, Canadian Institutes of Health Research, Pfizer Global Medical Grants, and St. Michael's Hospital Foundation. PJ and ACG are funded by the Canada Research Chairs Program.

Assessment of SARS-CoV-2 Seropositivity During the First and Second Viral Waves in 2020 and 2021 Among Canadian Adults.

Importance: The incidence of infection during SARS-CoV-2 viral waves, the factors associated with infection, and the durability of antibody responses to infection among Canadian adults remain undocumented. Objective: To assess the cumulative incidence of SARS-CoV-2 infection during the first 2 viral waves in Canada by measuring seropositivity among adults. Design, Setting, and Participants: The Action to Beat Coronavirus study conducted 2 rounds of an online survey about COVID-19 experience and analyzed immunoglobulin G levels based on participant-collected dried blood spots (DBS) to assess the cumulative incidence of SARS-CoV-2 infection during the first and second viral waves in Canada. A sample of 19 994 Canadian adults (aged ≥18 years) was recruited from established members of the Angus Reid Forum, a public polling organization. The study comprised 2 phases (phase 1 from May 1 to September 30, 2020, and phase 2 from December 1, 2020, to March 31, 2021) that generally corresponded to the first (April 1 to July 31, 2020) and second (October 1, 2020, to March 1, 2021) viral waves. Main Outcomes and Measures: SARS-CoV-2 immunoglobulin G seropositivity (using a chemiluminescence assay) by major geographic and demographic variables and correlation with COVID-19 symptom reporting. Results: Among 19 994 adults who completed the online questionnaire in phase 1, the mean (SD) age was 50.9 (15.4) years, and 10 522 participants (51.9%) were female; 2948 participants (14.5%) had self-identified racial and ethnic minority group status, and 1578 participants (8.2%) were self-identified Indigenous Canadians. Among participants in phase 1, 8967 had DBS testing. In phase 2, 14 621 adults completed online questionnaires, and 7102 of those had DBS testing. Of 19 994 adults who completed the online survey in phase 1, fewer had an educational level of some college or less (4747 individuals [33.1%]) compared with the general population in Canada (45.0%). Survey respondents were otherwise representative of the general population, including in prevalence of known risk factors associated with SARS-CoV-2 infection. The cumulative incidence of SARS-CoV-2 infection among unvaccinated adults increased from 1.9% in phase 1 to 6.5% in phase 2. The seropositivity pattern was demographically and geographically heterogeneous during phase 1 but more homogeneous by phase 2 (with a cumulative incidence ranging from 6.4% to 7.0% in most regions). The exception was the Atlantic region, in which cumulative incidence reached only 3.3% (odds ratio [OR] vs Ontario, 0.46; 95% CI, 0.21-1.02). A total of 47 of 188 adults (25.3%) reporting COVID-19 symptoms during phase 2 were seropositive, and the OR of seropositivity for COVID-19 symptoms was 6.15 (95% CI, 2.02-18.69). In phase 2, 94 of 444 seropositive adults (22.2%) reported having no symptoms. Of 134 seropositive adults in phase 1 who were retested in phase 2, 111 individuals (81.8%) remained seropositive. Participants who had a history of diabetes (OR, 0.58; 95% CI, 0.38-0.90) had lower odds of having detectable antibodies in phase 2. Conclusions and Relevance: The Action to Beat Coronavirus study found that the incidence of SARS-CoV-2 infection in Canada was modest until March 2021, and this incidence was lower than the levels of population immunity required to substantially reduce transmission of the virus. Ongoing vaccination efforts remain central to reducing viral transmission and mortality. Assessment of future infection-induced and vaccine-induced immunity is practicable through the use of serial online surveys and participant-collected DBS.

Predictors of self-reported symptoms and testing for COVID-19 in Canada using a nationally representative survey.

Random population-based surveys to estimate prevalence of SARS-CoV2 infection causing coronavirus disease (COVID-19) are useful to understand distributions and predictors of the infection. In April 2020, the first-ever nationally representative survey in Canada polled 4,240 adults age 18 years and older about self-reported COVID experience in March, early in the epidemic. We examined the levels and predictors of COVID symptoms, defined as fever plus difficulty breathing/shortness of breath, dry cough so severe that it disrupts sleep, and/or loss of sense of smell; and testing for SARS-CoV-2 by respondents and/or household members. About 8% of Canadians reported that they and/or one or more household members experienced COVID symptoms. Symptoms were more common in younger than in older adults, and among visible minorities. Overall, only 3% of respondents and/or household members reported testing for SARS-CoV-2. Being tested was associated with having COVID symptoms, Indigenous identity, and living in Quebec. Periodic nationally representative surveys of symptoms, as well as SARS-CoV-2 antibodies, are required in many countries to understand the pandemic and prepare for the future.

Resistance of colorectal cancer cells to 5-FUdR and 5-FU caused by Mycoplasma infection.

BACKGROUND: 5-Fluorouracil (5-FU) is an antineoplastic drug that targets thymidylate synthase (TS). Tumour cells can develop resistance to anti-TS drugs by a variety of mechanisms including up-regulation of TS protein and alterations in drug uptake and degradation. The possible mechanisms of the observed rapid development of resistance to the pyrimidine analogs 5-FUdR and 5-FU in cultured HCT116 colon cancer cells were investigated. MATERIALS AND METHODS: Cell survival was determined in resistant and control HCT116 cells treated with 5-FUdR and 5-FU for 7 days. The ability of the cells to take up and metabolize these drugs was determined by Western blotting and [3H]thymidine incorporation. RESULTS AND CONCLUSION: Resistant HCT116 cells were 5- and 100-fold more resistant to killing by 5-FU and 5-FUdR, respectively, than the parental cells and exhibited impaired uptake. Although the HCT116R cells were initially Mycoplasma free, a low level of Mycoplasma contamination was found in these cells after several weeks in culture. Sensitivity to 5-FUdR was restored by treatment with an anti-Mycoplasma antibiotic. Our observations emphasize the need for frequent testing for Mycoplasma contamination in any cell line under investigation for resistance to anti-TS drugs.

Nuclear expression of thymidylate synthase in colorectal cancer cell lines and clinical samples.

Thymidylate synthase (TS) [TYMS; OMIM reference number (188,350)] is normally considered to be a cytoplasmic enzyme. However, a few reports have suggested it may also be present in the nucleus. To explore this in more detail, we used a highly specific polyclonal antibody to TS and a combination of techniques, including immunocytochemistry, confocal microscopy, cell fractionation, and Western blotting. We developed cell line HeLa-55, a HeLa derivative that grossly overexpresses TS. Although the vast majority of TS was in the cytoplasm, some TS also was seen in the nucleus. TS in parental HeLa cells and in normal human fibroblasts was seen exclusively in the cytoplasm. HeLa-55 cells exposed to 5-fluorodeoxyuridine were fractionated and examined by Western blotting. Interestingly, both free TS and the ternary complex of TS were seen in the cytoplasmic fraction but only free TS was detected in the nuclear fraction. Amongst different cell lines examined, HCT-15 and normal fibroblasts showed no nuclear TS, HCC-2998 and SW-620 showed a small amount of nuclear TS, and HT-29, RKO, and HCT-116 showed a strong nuclear TS signal. Nuclear staining was clearly evident in some clinical colorectal specimens, both normal and malignant. This staining was definitively shown to be TS by competition with recombinant TS protein. A putative leucine-rich nuclear export sequence was identified but its function could not be confirmed. We conclude that small amounts of TS protein is present in the nucleus of some cell types but further work is needed to determine the significance of this observation.

Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative.

Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.

EGFR expression variance in paired colorectal cancer primary and metastatic tumors.

BACKGROUND: Previous studies indicate that drugs targeting the Epidermal Growth Factor Receptor (EGFR) signaling pathways can induce objective responses, prolong time to progression and improve survival of patients with metastatic colorectal cancer (mCRC). EGFR expression in the primary tumour may not predict response to these agents and data is conflicting regarding the correlation of EGFR expression in the primary tumour with the metastatic site. In other tumour sites, the presence of EGFR mutations was associated with efficacy in a subset of patients. OBJECTIVES: The goal of this study is to correlate tumour EGFR expression between primary and liver metastatic sites, and to assess the mutational status in the EGFR kinase domain. METHODS: This is a single center retrospective study of patients who underwent surgical resection of CRC, for whom paired paraffin-embedded tissue blocks of primary tumours and resected liver metastases were available. EGFR immunostaining and mutation analyses were preformed. RESULTS: Fifty six paired colorectal primaries and metastases were available for analysis. EGFR was detectable in 96.6% of the primary samples and in 89.7% of the metastatic samples. Perfect concordance in the intensity score between the primary and the metastases was found in 46.5% of the cases. While individual pairs were poorly concordant for intensity, the proportion of primaries with intense staining was similar to the proportion with intense staining in the metastatic samples. Overall survival did not correlate with either EGFR expression in the primary tumour, or with EGFR expression in the metastasis. There were 2 cases with mutations in the EGFR kinase domain. Both mutations were found in exon21 C>T. CONCLUSIONS: In this analysis, EGFR expression in the primary tumor site was not predictive of its level in the metastasis. EGFR expression levels in the primaries and in the metastases do not appear to be useful prognostic markers.

The role of a formaldehyde dehydrogenase-glutathione pathway in protein S-nitrosation in mammalian cells.

Intracellular sulfhydryls, both protein and non-protein, are potential targets of nitric oxide-related species. S-Nitrosation of proteins can occur in vivo and can affect their activity. Metabolic pathways that regulate protein S-nitrosation are therefore likely to be biologically important. We now report that formaldehyde dehydrogenase, an enzyme that decomposes S-nitrosoglutathione, can indirectly regulate the level of cellular protein S-nitrosation. Nitrogen oxide donors induced high levels of protein S-nitrosation in HeLa cells and lower levels in Mutatect fibrosarcoma cells, as determined by Saville-Griess assay and Western-dot-blot analysis. Depletion of glutathione by treatment with buthionine sulfoximine markedly increased protein S-nitrosation in both cell lines. Glutathione depletion also increased cytokine-induced S-nitrosation in brain endothelial cells. Formaldehyde dehydrogenase activity was 2-fold higher in Mutatect than in HeLa cells. We downregulated formaldehyde dehydrogenase activity in Mutatect cells by stably expressing antisense RNA and short-interfering RNA. In these cells, both protein S-nitrosation and S-nitrosoglutathione levels were significantly enhanced after exposure to nitrogen oxide donors as compared to parental cells. Overall, a strong inverse correlation between total S-nitrosothiols and formaldehyde dehydrogenase activity was seen. Inhibition of glutathione reductase, the enzyme that converts oxidized to reduced glutathione, by dehydroepiandrosterone similarly increased protein S-nitrosation and S-nitrosoglutathione levels in both cell lines. Our results provide the first evidence that formaldehyde dehydrogenase-dependent decomposition of S-nitrosoglutathione plays a role in protecting against nitrogen oxide-mediated protein S-nitrosation. We propose that formaldehyde dehydrogenase and glutathione reductase participate in a glutathione-dependent metabolic cycle that decreases protein S-nitrosation following exposure of cells to nitric oxide.

Dietary vitamin E affects neutrophil distribution and genetic instability in murine Mutatect tumors.

Vitamin E is best known for its ability to scavenge reactive oxygen and nitrogen species. Solid tumors are frequently infiltrated with leukocytes, a potential source of these reactive species. The Mutatect tumor model is a fibrosarcoma that can be grown subcutaneously in syngeneic C57BL/6 mice. We previously showed that these tumors are infiltrated with neutrophils and that the number of neutrophils correlates with the number of hypoxanthine phosphoribosyl transferase (hprt) mutations and loss of an interleukin-8 (IL-8) transgene. Neutrophils are a source of nitric oxide, and tumors contain nitrotyrosine, a marker of damage by nitric oxide-related species. We also showed previously that dietary vitamin E supplements markedly lower the frequency of hprt mutants and the level of myeloperoxidase (a neutrophil marker) in a tumor fraction containing "loosely bound" cells. In the present report, we examine the effect of dietary vitamin E in greater detail. No effect on inducible nitric oxide synthase expression or nitrotyrosine levels was observed. However, dietary vitamin E induced a major redistribution of neutrophils from the loosely bound cellular fraction to the "stromal" fraction, while the total number of neutrophils in tumors was essentially unchanged. The loss of the IL-8 transgene seen earlier in Mutatect tumors was largely prevented. Vitamin E also prevented the large increase in hprt mutants (in the cellular and stromal fractions). Thus vitamin E appears to be protective against genotoxicity by scavenging reactive species, but also its ability to affect the distribution of neutrophils within tumors may be important.

Selective nitration of histone tyrosine residues in vivo in mutatect tumors.

Nitric oxide-derived reactive species have been implicated in many disorders. Protein nitrotyrosine is often used as a stable marker of these reactive species. Using immunohistochemistry, we have previously detected nitrotyrosine in murine Mutatect tumors, where neutrophils are the principal source of nitric oxide. We now report on the identification of several prominent nitrotyrosine-containing proteins. Using Western blot analysis, nitrotyrosine in higher molecular mass proteins (>20 kDa) was detected in tumors containing a high number of neutrophils but not in tumors with fewer neutrophils. Staining for nitrotyrosine was consistently seen in low molecular mass proteins (< or =15 kDa), regardless of the level of neutrophils. Protein nitrotyrosine was not seen in Mutatect cells growing in vitro. Treatment with nitric oxide donors produced nitration of < or =15-kDa proteins, but only after extended periods. These small proteins, both from tumors and cultured cells, were identified by mass spectrometry to be histones. Only a subset of tyrosine residues was nitrated. Selective nitration may reflect differential accessibility of different tyrosine residues and the influence of neighboring residues within the nucleosome. The prominence of histone nitration may reflect its relative stability, making this post-translational modification a potentially useful marker of extended exposure of cells or tissues to nitric oxide-derived reactive species.

Distribution of protein nitrotyrosine in synovial tissues of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: Because nitric oxide related species have been found in the inflamed joints of patients with arthritis, we investigated whether protein nitrotyrosine (a marker of tissue exposure to peroxynitrite) is present in their synovial tissues. METHODS: Protein nitrotyrosine was detected immunohistochemically and by Western blot analysis. Synovial tissues removed surgically from 12 patients with rheumatoid arthritis (RA) (mean age 63.7 yrs) and 20 with osteoarthritis (OA) (mean age 66.6 yrs) were studied. RESULTS: Nitrated proteins were detected immunohistochemically in all of 18 tissues examined. Diffuse staining of the stroma was seen in all patients, with more extensive staining in RA than OA (p = 0.008). Intense staining was detected in some lymphocytes, but not in others, even within a single lymphoid aggregate. Neutrophils did not stain for nitrotyrosine. Vascular endothelial cells stained for nitrotyrosine but adjoining smooth muscle cells did not. Both cytoplasmic and nuclear staining was seen in macrophages, endothelial cells, and lymphocytes. Numerous bands of nitrated proteins were detected by Western blot analysis of 15 synovial tissue extracts. Inducible nitric oxide synthase (iNOS) was detected immunohistochemically in endothelial cells, macrophages, vascular smooth muscle cells, and synoviocytes. CONCLUSION: Nitrotyrosine-containing proteins were found in essentially all synovia from RA and OA patients. The most prominent site of nitration in all cases was the stroma. iNOS, the likely source of the nitrating species, was found in a variety of cell types.

Cutting edge: MHC class II-restricted peptides containing the inflammation-associated marker 3-nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune response.

Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-E(K)-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC(88-103)) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC(88-103) with nitrotyrosine abrogates recognition by the MCC(88-103)-specific T cell hybridoma 2B4. CBA (H2(K)) mice immunized with MCC(88-103) or nitrated MCC(88-103) peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC(88-103), but exhibited a robust immune response against nitrated MCC(88-103). Analysis of T cell hybridomas specific for nitrated-MCC(88-103) indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.

Dose-dependent effects of dietary alpha- and gamma-tocopherols on genetic instability in mouse Mutatect tumors.

Vitamin E in foodstuffs is a mixture of tocopherols. In mouse Mutatect tumors, a model designed to detect DNA mutations, the hypoxanthine phosphoribosyltransferase (Hprt) gene mutation frequency is associated with the number of tumor-infiltrating neutrophils and both are markedly decreased in mice fed high levels of alpha-tocopherol. Dietary alpha-tocopherol is also associated with a decrease in neutrophil-associated loss of an interleukin 8 (IL-8)-expressing transgene in this tumor model. We examined Hprt gene mutation frequency (expressed as the number of 6-thioguanine-resistant colonies per 10(5) clonable tumor cells), IL-8 transgene loss, and myeloperoxidase activity (an indirect measure of neutrophil number) in tumors from Mutatect mice fed diets supplemented with various concentrations of D-alpha-tocopherol acetate and/or D-gamma-tocopherol acetate or neither tocopherol for 4 weeks. Hprt gene mutation frequency and myeloperoxidase activity were statistically significantly lower in tumor cells from mice fed alpha-tocopherol at 50 or 100 mg/kg body weight per day than in tumor cells from mice fed 0 mg/kg body weight per day alpha-tocopherol (P<.001 for each comparison). IL-8 transgene loss occurred in 28 of 28 tumors (100%; 95% confidence interval [CI] = 86% to 100%) from mice fed alpha-tocopherol at 50 mg or less/kg body weight per day and seven of 18 tumors (39%; 95% CI = 24% to 54%) from mice fed 100 mg/kg body weight per day (P<.001, Fisher's exact test, referent groups [pooled] 0, 25, and 50 mg/kg). gamma-Tocopherol had no detectable effect on any of the three endpoints. Thus, dietary alpha-tocopherol decreases two forms of genetic instability in a dose-dependent manner in this experimental tumor model.