Introduction During initial stages of the pandemic, SARS-CoV-2 infection during pregnancy was related to adverse pregnancy outcomes and alterations in the placenta. Whether placental abnormalities in pregnant women with COVID-19 still persist afterwards remains poorly studied. Here, we determined whether the absence of obstetric complications after maternal COVID-19 disease, including preeclampsia, is accompanied by a complete return to normalcy in terms of placental physiology. Methods Placental RNA was purified from placental samples from SARS-CoV-2 positive mothers taken either in 2022, when Omicron was the predominant variant of concern (termed Omicron) (n = 21); or from healthy pregnancies predating the pandemic (termed preCOVID-19). Our cohort included samples from pregnant women who got infected weeks and even months before term. We performed RNA-seq, identified differentially expressed genes and examined to which biological, biochemical and cellular pathways they belong, using gene set enrichment analysis. Results We identified 71 differentially expressed genes (DEGs) (defined by p-value ≤0.05 and fold change (FC) of ≤ -2 or ≥2). The alterations identified in placentas delivered by mothers who suffered an episode of COVID-19 disease could be mainly attributed to pathways related to organogenesis, extracellular matrix organization and oxygen transport. These alterations were also detected after exclusion of gestational diabetes mellitus (GDM) samples. Although none of the samples were taken from cases of preeclampsia, several of the relevant DEGs have been previously reported as dysregulated in hypertensive disorders of pregnancy including preeclampsia. Discussion We propose that maternal SARS-CoV-2 infection causes alterations in gene expression that are indicative of vascular defects in the placenta.

Retroviral reverse transcriptase activity and the increased expression of human endogenous retroviruses (HERVs) are associated with amyotrophic lateral sclerosis (ALS). We were interested in confirming HERVK overexpression in the ALS brain, its use as an accessory diagnostic marker for ALS, and its potential interplay with neuroinflammation. Using qPCR to analyze HERVK expression in peripheral blood mononuclear cells (PBMCs) and in postmortem brain samples from ALS patients, no significant differences were observed between patients and control subjects. By contrast, we report alterations in the expression patterns of specific HERVK copies, especially in the brainstem. Out of 27 HERVK copies sampled, the relative expression of 17 loci was >1.2-fold changed in samples from ALS patients. In particular, the relative expression of two HERVK copies (Chr3-3 and Chr3-5) was significantly different in brainstem samples from ALS patients compared with controls. Further qPCR analysis of inflammation markers in brain samples revealed a significant increase in NLRP3 levels, while TNFA, IL6, and GZMB showed slight decreases. We cannot confirm global HERVK overexpression in ALS, but we can report the ALS-specific overexpression of selected HERVK copies in the ALS brain. Our data are compatible with the requirement for better patient stratification and support the potential importance of particular HERVK copies in ALS.

Abstract Objectives Myocardial infarction evokes inflammation and activation of quiescent cardiac fibroblasts, leading to heart failure progression. We hypothesized that targeted molecular imaging of fibroblast activation protein (FAP) on activated fibroblasts could predict subsequent remodeling and monitor fibroblast response to anti-inflammatory therapy. Methods In vitro uptake of 68Ga-FAPI-46 was assessed in human cardiac fibroblasts stimulated by supernatant from inflammatory macrophages. C57Bl/6 mice underwent 60min left coronary artery ischemia/ reperfusion (I/R) (n=22) or sham (n=7). Serial 68Ga-FAPI-46 and 18F-FDG PET imaging was performed 1d, 3d, 7d, 14d and 42d after surgery. Left ventricle (LV) function and perfusion defect size were assessed by MRI and SPECT at 6wks. Additional animals were treated with anti-inflammatory enalapril and similarly scanned. Results Exposure to M1-like macrophage supernatant increased 68Ga-FAPI46 uptake by cardiac fibroblasts (p=0.01), associated with higher expression of IL-1β (p<0.05) and TGF-β (p=0.003). I/R induced moderate infarct size at 6wks post I/R (18.1±5.3% of LV). I/R animals exhibited increased LV volumes compared to sham (systole, 38±7 vs 21±4µL, p<0.001; diastole, 69±9 vs 49±4µL, p<0.001) and decreased LV ejection fraction (EF)(46±10 vs 58±2%, p<0.001). Serial FAP imaging revealed biphasic upregulation in the infarcted myocardium. Signal intensity 3d after injury was significantly increased (%ID/g, 0.69.±0.1 vs 1.3±0.3, p<0.001). After declining to sham levels at 7d, signal was again elevated 14d after I/R(%ID/g 0.70±0.1 vs 0.94±0.1, p=0.05) before returning to sham levels at 6wks. Signal increase was not restricted to infarct area, but similarly observed in non-infarcted remote myocardium at 3d (p<0.001) and 14d (p<0.05), suggesting involvement of both replacement and reactive fibrosis. There is modest inverse correlation between 3d infarct area FAPI signal and LVEF 6wks later (r=-0.43, p<0.05), but stronger with 14d FAPI signal (r=-0.51, p<0.05). Assuming the elevation at 3d would reflect inflammatory cell infiltration and lead to replacement fibrosis, whereas the later wave would derive from interstitial fibrosis, we treated animals with ACE inhibitor enalapril for 10d, beginning 2d prior to surgery to prevent mobilization of myeloid cells from the spleen. FAPI signal was significantly reduced in the infarct area in enalapril-treated mice at 3d (p=0.05), supporting the notion that early FAP expression is regulated by inflammatory environment. Conclusions Molecular imaging of FAP provides a prognostic biomarker of subsequent remodeling reflecting both early inflammation driven replacement and later interstitial fibrosis, ultimately supporting its capacity to monitor novel therapies.

The original SARS-CoV-2 lineages have been replaced by successive variants of concern (VOCs) over time. The aim of this study was to perform an assessment of the placental infection by SARS-CoV-2 according to the predominant variant at the moment of COVID-19 diagnosis. This was a prospective study of SARS-CoV-2-positive pregnant women between March 2020 and March 2022. The population was divided into pregnancies affected by COVID-19 disease during 2020 (Pre-VOC group) and pregnancies affected after December 2020 by SARS-CoV-2 variants of concern (VOC group). The presence of virus was assessed by RT-PCR, and the viral variant was determined by whole genome sequencing. A total of 104 placentas were examined, among which 54 cases belonged to the Pre-VOC group and 50 cases belonged to the VOC group. Sixteen positive placental RT-PCR tests for SARS-CoV-2 were reported. The NGS analysis confirmed the SARS-CoV-2 lineage in placenta tissue. All samples corresponded to the Pre-VOC group, whereas no placental presence of SARS-CoV-2 was detected in the VOC group (16, 29.6% vs. 0, 0.0% p = 0.000). Preterm birth (9, 16.7% vs. 2, 4%; p = 0.036) and hypertensive disorders of pregnancy (14, 25.9% vs. 3, 6%; p = 0.003) were more frequent in the Pre-VOC group than in the VOC group. Finally, the VOC group was composed of 23 unvaccinated and 27 vaccinated pregnant women; no differences were observed in the sub-analysis focused on vaccination status. In summary, SARS-CoV-2-positive placentas were observed only in pregnancies infected by SARS-CoV-2 wildtype. Thus, placental SARS-CoV-2 presence could be influenced by SARS-CoV-2 variants, infection timing, or vaccination status. According to our data, the current risk of SARS-CoV-2 placental infection after maternal COVID disease during pregnancy should be updated.

and IntroductionHuman endogenous retrovirus (HERV) are the present day versions of retroviral germline infections that have occured millions of years ago, which occupy about 8 % of the genome [1]. While they are mostly replication deficient, they are known to express RNA and protein [2] during particular developmental stages, or as a response to aging [3], inflammation and a wide range of pathologies [4]. A human retrovirus discovered in Multiple Sclerosis (MS) patients [5], turned out to be the prototype of a novel HERV family referred to as HERVW [6]. The HERVW family consists of 213 elements, 12 out of which are complete proviral copies with intact LTRs [7]. Increased expression of HERVW in peripheral blood mononuclear cells (PBMCs) has been repeatedly associated with MS, and the presence of HERVW protein or elevated RNA transcription has been correlated with disease activity [8,9,10]. While a contribution of HERVW-encoded proteins to brain disease is suggested by their presence in MS-associated brain lesions, expression in peripheral organs may be involved in the disease process through cytokine-induced damage to the blood brain barrier and subsequent infiltration of monocytes. Alterations in peripheral expression may also serve as a useful and practical marker for the diagnostics of this CNS disease. Therefore, we quantified overall HERVW levels and identified individual HERVW loci actually transcribed in PBMCs. Analysis was carried out in patients diagnosed with Clinically Isolated Syndrome (CIS), a precursor to MS, defined by a single episode of neurologic symptoms lasting at least 24 h. CIS is an indicator of future development of MS, as 60 % of the people diagnosed with CIS develop MS [11]. These patients potentially represent the earliest stage of MS routinely available for clinical analysis. We undertook a Next Generation Sequencing (NGS)-based analysis of transcripts amplified from cDNA obtained from patients with CIS and samples from healthy controls. Data presented from this pilot experiment indicate that the relative frequency of specific HERVW copies is altered in PBMC of CIS patients, even in the absence of overall HERVW overexpression. Such altered frequency appears to be derived from less abundantly transcribed but potentially MS-related HERVW loci.

Introduction: Studies described an increased frequency of hypertensive disorders of pregnancy after a COVID-19 episode. There is limited evidence about SARS-CoV-2 viral load in placenta. This study aimed to investigate the relationship between SARS-CoV-2 viral load in placenta and clinical development of HDP after COVID-19 throughout different periods of gestation. Methods: This was a case-control study in women with and without gestational hypertensive disorders (HDP) after SARS-CoV-2 infection diagnosed by RT-PCR during pregnancy. Patients were matched by gestational age at the moment of COVID-19 diagnosis. We performed an analysis of SARS-CoV-2 RNA levels in placenta. Results: A total of 28 women were enrolled. Sixteen patients were diagnosed with COVID-19 during the third trimester and the remaining twelve patients in the others trimesters. Ten placentas (35.7%) were positive for SARS-CoV-2, nine of them (90%) belonged to the HDP group versus one (10%) in control group (p=0.009). Those cases with the highest loads of viral RNA developed severe-preeclampsia. Conclusion: The presence of SARS-CoV-2 was more frequent in placentas of patients with HDP after COVID-19. There seems to be a relationship between high viral load in the placenta and the development of hypertensive disorders. We found SARS-CoV-2 viral load in placenta after birth in mothers infected at the first half of pregnancy, but with negative nasopharyngeal RT-PCR at delivery. Our data suggest that SARS-CoV-2 infection during pregnancy could trigger gestational hypertensive disorders through placenta-related mechanisms.

Introduction: Studies described an increased frequency of hypertensive disorders of pregnancy (HDP) after a COVID-19 episode. There is limited evidence about SARS-CoV-2 viral load in placenta. This study aimed to investigate the relationship between SARS-CoV-2 viral load in the placenta and clinical development of HDP after COVID-19 throughout different periods of gestation. Methods: This is a case-control study in women with and without gestational hypertensive disorders after SARS-CoV-2 infection diagnosed by RT-PCR during pregnancy. Patients were matched by gestational age at the moment of COVID-19 diagnosis. We performed an analysis of SARS-CoV-2 RNA levels in placenta. Results: A total of 28 women were enrolled. Sixteen patients were diagnosed with COVID-19 during the third trimester and the remaining 12 patients in the other trimesters. Ten placentas (35.7%) were positive for SARS-CoV-2, 9 of them (9/14, 64.3%) belonged to the HDP group versus 1 (1/14, 7.2%) in the control group (p = 0.009). Those cases with the highest loads of viral RNA developed severe preeclampsia (PE). Conclusion: Among women diagnosed with COVID-19 during pregnancy, the presence of SARS-CoV-2 in the placenta was more frequent among women suffering from PE or gestational hypertension. Furthermore, the most severe cases of HDP were associated with high placental viral load, not necessarily associated with a positive nasopharyngeal RT-PCR at delivery. Our data suggest that SARS-CoV-2 infection during pregnancy could trigger gestational hypertensive disorders through persistent placental infection and resulting placental damage.

Aim: The aim of this study was to determine if alterations in DNA methylation in the human placenta would support suspected preterm labor as a pathologic insult associated with diminished placental health. Methods: We evaluated placental DNA methylation at seven loci differentially methylated in placental pathologies using targeted bisulfite sequencing, in placentas associated with preterm labor (term birth after suspected preterm labor [n = 15] and preterm birth [n = 15]), and controls (n = 15). Results: DNA methylation levels at the NCAM1 and PLAGL1 loci in placentas associated with preterm labor did differ significantly (p < 0.05) from controls. Discussion: Specific alterations in methylation patterns indicative of an unfavourable placental environment are associated with preterm labor per se and not restricted to preterm birth.

p16 hypermethylation in Barrett's carcinogenesis has been evaluated in studies which did not take into account sample heterogeneity and yielded qualitative (methylated/unmethylated) instead of accurate quantitative (percentage of CpG methylation) data. We aimed to measure the degree of p16 methylation in pure samples representing all the steps of Barrett's tumorogenesis and to evaluate the influence of sample heterogeneity in methylation analysis. Methods: 77 paraffin-embedded human esophageal samples were analyzed. Histological grading was established by two pathologists in: negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Areas of interest were selected by laser-capture microdissection. p16 methylation was quantified by pyrosequencing. An adjacent section of the whole sample was also analyzed to compare methylation data. Results: After microdissection, we obtained 15 samples of squamous epithelium, 36 non-dysplastic Barrett's esophagus, 3 indefinite for dysplasia, 24 low-grade dysplasia, 4 high-grade dysplasia and 12 adenocarcinoma. Squamous epithelium showed the lowest methylation rates: 6% (IQR 5-11) vs. 11%(7-39.50) in negative/indefinite for dysplasia, p < 0.01; 10.60%(6-24) in low-grade dysplasia, p < 0.05; and 44.50%(9-66.75) in high-grade dysplasia/adenocarcinoma, p < 0.01. This latter group also exhibited higher methylation rates than Barrett's epithelium with and without low-grade dysplasia (p< 0.05). p16 methylation rates of microdissected and non-microdissected samples did not correlate unless the considered histological alteration comprised > 71% of the sample. Conclusions: p16 methylation is an early event in Barrett's carcinogenesis which increases with the severity of histological alteration. p16 methylation rates are profoundly influenced by sample heterogeneity, so selection of samples is crucial in order to detect differences.

The cancer stem cell (CSC) model suggests that there are subsets of cells within a tumor with increased proliferation and self-renewal capacity, which play a key role in therapeutic resistance. The importance of cyclooxygenase-2 (COX-2) in carcinogenesis has been previously established and the use of COX-2 inhibitors as celecoxib has been shown to exert antitumor effects. The present study investigated whether treatment of esophageal adenocarcinoma (EAC) cells with 5-fluorouracil (5-FU) or the growth of tumor spheres increased the proportion of CSCs and also if treatment with celecoxib was able to reduce the putative CSC markers in this tumor. OE19 and OE33 EAC cells surviving 5-FU exposure exhibited an increase in CSC markers CD24 and ABCG2 and also an increased resistance to apoptosis. EAC cell lines had the capacity to form multiple spheres displaying typical CSC functionalities such as self-renewal and increased CD24 levels. In addition, after the induction of differentiation, cancer cells reached levels of CD24 similar to those observed in the parental cells. Treatment with celecoxib alone or in combination with 5-FU also resulted in a reduction of CD24 expression. Moreover, celecoxib inhibited the growth of tumor spheres. These findings showing a reduction in CSC markers induced by celecoxib suggest that the COX-2 inhibitor might be a candidate for combined chemotherapy in the treatment of EAC. However, additional clinical and experimental studies are needed.

Our aim was to evaluate whether placental subclinical inflammation biomarkers predict impaired cognitive development in two year-old children after an episode of suspected preterm labour. Two-year-old children who were born late preterm (n=21) or at term after suspected preterm labour (n=16) were compared with at-term control children (n=10). Placental samples were collected immediately after delivery. We quantitatively analysed the mRNA expression of inflammatory markers (IL6, IFNγ, and TNFα). Placental inflammation was defined as the presence of both IL6 and TNFα mRNA expression were over 1.5 MoMs. Neurodevelopment was evaluated at a corrected age of 24-29 months with the use of the Merrill-Palmer-Revised Scales of Development. Inflammation was present in the 10% of the controls, 14.3% of late preterm and 18.8% of term-born after suspected preterm labour placentas. Both term-born and late preterm two year-old infants with signs of subclinical placental inflammation had significantly lower mean scores on global cognitive index (86.71 vs 98.29; p= 0.014), cognition (86.85 vs 97.86; p=0.019), fine motor (89.42 vs 97.08; p=0.08), receptive language (83.28 vs 97.56; p=0.08), active and diligent (74 vs 99.72; p=0.07), and easy temper style (81.71 vs 93.62; p=0.06) subtest. An increased placental mRNA expression of IL6 was correlated with the active and diligent (-0.328; p=0.024) and angry and unhelpful (0.302; p=0.039) subtest. Whereas, an increased mRNA expression of TNF-alfa was also correlate with the cognition (-0.294; p=0.047), adaptive behaviour and self-care (-0.353; p=0.015), fine motor (-0.316; p=0.033), receptive language (-0.294; p=0.047), expressive language (-0.310; p=0.036), active and diligent (-0.310; p=0.034), temperament (-0.306; p=0.038) subtest. Placental inflammation is a risk factor for impaired cognitive development at early childhood age after suspected preterm labour both in term and preterm neonates.

Variations in DNA repair genes have been reported as key factors in gastric cancer (GC) susceptibility but results among studies are inconsistent. We aimed to assess the relevance of DNA repair gene polymorphisms and environmental factors to GC risk and phenotype in a Caucasian population in Spain. Genomic DNA from 603 patients with primary GC and 603 healthy controls was typed for 123 single nucleotide polymorphisms in DNA repair genes using the Illumina platform. Helicobacter pylori infection with CagA strains (odds ratio (OR): 1.99; 95% confidence interval (CI): 1.55-2.54), tobacco smoking (OR: 1.77; 95% CI: 1.22-2.57), and family history of GC (OR: 2.87; 95% CI: 1.85-4.45) were identified as independent risk factors for GC. By contrast, the TP53 rs9894946A (OR: 0.73; 95% CI: 0.56-0.96), TP53 rs1042522C (OR: 0.76; 95% CI: 0.56-0.96), and BRIP1 rs4986764T (OR: 0.55; 95% CI: 0.38-0.78) variants were associated with lower GC risk. Significant associations with specific anatomopathological GC subtypes were also observed, most notably in the ERCC4 gene with the rs1799801C, rs2238463G, and rs3136038T variants being inversely associated with cardia GC risk. Moreover, the XRCC3 rs861528 allele A was significantly increased in the patient subgroup with diffuse GC (OR: 1.75; 95% CI: 1.30-2.37). Our data show that specific TP53, BRIP1, ERCC4, and XRCC3 polymorphisms are relevant in susceptibility to GC risk and specific subtypes in Caucasians.

INTRODUCTION:Suspected preterm labour occurs in around 9% of pregnancies. However, almost two-thirds of women admitted for threatened preterm labour ultimately deliver at term and are considered risk-free for fetal development.METHODS:We examined placental and umbilical cord blood samples from preterm or term deliveries after threatened preterm labour as well as term deliveries without threatened preterm labour. We quantitatively analysed the mRNA expression of inflammatory markers (IL6, IFNγ, and TNFα) and modulators of angiogenesis (FGF2, PGF, VEGFA, VEGFB, and VEGFR1).RESULTS:A total of 132 deliveries were analysed. Preterm delivery and term delivery after suspected preterm labour groups showed similar increases in TNFα expression compared with the term delivery control group in umbilical cord blood samples. Placental samples from preterm and term deliveries after suspected preterm labour exhibited significantly increased expression of TNFα and IL6 and decreased expression of IFNγ. Suspected preterm labour was also associated with altered expression of angiogenic factors, although not all differences reached statistical significance.DISCUSSION:We found gene expression patterns indicative of inflammation in human placentas after suspected preterm labour regardless of whether the deliveries occurred preterm or at term. Similarly, a trend towards altered expression of angiogeneic factors was not limited to preterm birth. These findings suggest that the biological mechanisms underlying threatened preterm labour affect pregnancies independently of gestational age at birth.

Two recent genome‐wide association studies in Asians have reported the association between the PSCA (prostate stem cell antigen) rs2294008C>T gene polymorphism and two Helicobacter pylori infection‐related diseases such as gastric cancer (GC) and duodenal ulcer (DU). Since rs2294008 allele frequencies differ notably among ethnicities, we aimed to assess the role of rs2294008 on the susceptibility to GC and DU in a Caucasian population in Spain. Moreover, the relevance of rs2294008 on GC prognosis was evaluated. Genomic DNA from 603 Spanish patients with primary GC, 139 with DU and 675 healthy controls was typed for the PSCA rs2294008C>T polymorphism by PCR‐TaqMan assays. H. pylori infection [odds ratio (OR): 8.27; 95% confidence interval (CI): 3.45–15.33] and nonsteroidal anti‐inflammatory drugs (OR: 6.54; 95% CI: 3.19–12.43) were identified as independent risk factors for DU whereas the rs2294008T allele was associated with reduced risk of developing the disease (OR: 0.52; 95% CI: 0.33–0.82). Infection with CagA strains (OR: 2.10; 95% CI: 1.63–2.34), smoking (OR: 1.93; 95% CI: 1.54–2.61), family history of GC (OR: 2.83; 95% CI: 2.01–3.83), and the rs2294008T allele (OR: 1.46; 95% CI: 1.07–1.99) were associated with increased risk of GC. Interestingly, the association with the rs2294008T allele was restricted to noncardia GC (OR: 1.43; 95% CI: 1.12–1.82), particularly of the diffuse histotype (OR: 1.59; 95% CI: 1.16–1.92). Finally, Cox regression analysis identified the rs2294008T variant as a prognosis factor associated with worse overall survival in patients with diffuse‐type GC (hazard ratio: 1.85; 95% CI: 1.12–3.06). From these results we conclude that the PSCA rs2294008 polymorphism is involved in the susceptibility to GC and DU, as well as in the prognosis of the diffuse‐type of GC in Caucasians.

Colorectal cancer (CRC) screening has been shown to reduce CRC incidence and mortality through the endoscopic detection and removal of colorectal adenomas.Still, these patients are at increased risk for developing metachronous adenomas or even cancer, with the recurrence rate reaching the 50%.The pleiotropic effects of higher levels of PGE 2 contribute to key steps of cancer development, including cell proliferation, angiogenesis, invasiveness and migration, inhibition of apoptosis and immunosurveillance as a refletion of deregulation of ATP-binding cassete sub-family c member 4 (ABCC4) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1) genes responsable for carrying PGE 2 accross the membrane.To evaluate the influence of genetic polymorphisms in ABCC4 and SLCO2A1 on the risk and time for colorectal adenoma recurrence a retrospective case-cohort study was designed gathering 195 patients diagnosed with colorectal adenomas.Adenoma reccurence was defined has the diagnosis of an adenoma after a total normal colonoscopy at least one year after the initial diagnosis.Thirty-three tagSNPs were characterized using the MassARRAY iPLEX Gold technology based on multiplex amplification followed by mass-spectrometric product separation.Three tagSNPs were identified as susceptibility biomarkers for colorectal adenoma recurrence after a bootstrap analysis.The rs1131598GG homozygous genotype of SLCO2A1 gene was associated with an enhanced risk of 6.3 (95%CI:1.31-30.0,P=0.021).In contrast and under a dominant model of inheritance, the rs1751031 and rs9524821 polymorphisms in ABCC4 gene displayed a protective behaviour (OR=0.29,95%CI:0.12-0.72,P=0.007 and OR=0.42, 95%CI:0.19-0.93,P=0.033, respectively).Furthermore, when stratifying patients considering the endoscopic findings at baseline colonoscopy, low-risk individuals carriers of rs2274403AA genotype in ABCC4 gene had a lower interval until recurrence (85 (29-140) vs 122 (109-135), P=0.011) with 44% of metachronous tumors developing by 36 months (vs 23% for AG/GG).This study demonstrates for the first time the involvement of genetic variants in PGE 2 transporters in colorectal adenoma recurrence.The incorporation of genetically-based approaches might allow an optimization of current risk models for the development of metachronous colorectal adenomas or even more advanced lesions possible laeding to a decrease in CRC burden and mortality.