Progesterone promotes Cxcl2-dependent vaginal neutrophil killing by activating cervical resident macrophage-neutrophil crosstalk.

Vaginal infections in women of reproductive age represent a clinical dilemma with significant socio-economic implications. The understanding of mucosal immunity failure during early pathogenic invasions that allow the pathogen to grow and thrive is far from complete. Neutrophils infiltrate most tissues following circadian patterns as part of normal repair, regulation of microbiota, or immune surveillance and become more numerous after infection. Neutrophils are responsible for maintaining vaginal immunity. Specific to the vagina, neutrophils continuously infiltrate at high levels, although during ovulation they retreat to avoid sperm damage and permit reproduction. Here we show that after ovulation, progesterone promotes resident vaginal macrophage-neutrophils crosstalk by up-regulating Yolk sac and early fetal organs (Folr2+ (folate receptor 2)) macrophage Cxcl2 expression, in a Tnfa-monocyte-derived macrophage (Cx3cr1hi MHCIIhi) mediated manner, to activate neutrophils' capacity to eliminate sex-transmitted and opportunistic microorganisms. Indeed, progesterone plays an essential role in conciliating the balance between the commensal microbiota, sperm, and the threat of pathogens: because progesterone not only promotes a flurry of neutrophils but also increases neutrophilic fury to restore immunity after ovulation to thwart pathogenic invasion post-intercourse. Therefore, modest progesterone dysregulations could lead to a suboptimal neutrophilic response, resulting in insufficient mucosal defense and recurrent unresolved infections.

The Effect of Polymorphisms and Other Biomarkers on Infliximab Exposure in Paediatric Inflammatory Bowel Disease: Development of a Population Pharmacokinetic Model.

BACKGROUND: Therapeutic drug monitoring (TDM) of infliximab has been shown to be a effective strategy for inflammatory bowel disease (IBD). Population pharmacokinetic (PopPK) modeling can predict trough concentrations for individualized dosing. OBJECTIVE: The aim of this study was to develop a PopPK model of infliximab in a paediatric population with IBD, assessing the effect of single nucleotide polymorphisms (SNPs) and other biomarkers on infliximab clearance. METHODS: This observational and ambispective single-centre study was conducted in paediatric patients with IBD treated with infliximab between July 2016 and July 2022 in the Paediatric Gastroenterology Service of the Hospital Universitari Vall d'Hebron (HUVH) (Spain). Demographic, clinical, and analytical variables were collected. Twenty SNPs potentially associated with variations in the response to infliximab plasma concentrations were analysed. infliximab serum concentrations and antibodies to infliximab (ATI) were determined by ELISA. PopPK modelling was performed using nonlinear mixed-effects analysis (NONMEM). RESULTS: Thirty patients (21 males) were included. The median age (range) at the start of infliximab treatment was 13 years (16 months to 16 years). A total of 190 samples were obtained for model development (49 [25.8%] during the induction phase). The pharmacokinetics (PK) of infliximab were described using a two-compartment model. Weight, erythrocyte sedimentation rate (ESR), faecal calprotectin (FC), and the SNP rs1048610 (ADAM17) showed statistical significance for clearance (CL), and albumin for inter-compartmental clearance (Q). Estimates of CL1 (genotype 1-AA), CL2 (genotype 2-AG), CL3 (genotype 3-GG), Q, Vc, and Vp (central and peripheral distribution volumes) were 0.0066 L/h/46.4 kg, 0.0055 L/h/46.4 kg, 0.0081 L/h/46.4 kg, 0.0029 L/h/46.4 kg, 0.6750 L/46.4 kg, and 1.19 L/46.4 kg, respectively. The interindividual variability (IIV) estimates for clearance, Vc, and Vp were 19.33, 16.42, and 36.02%, respectively. CONCLUSIONS: A popPK model utilising weight, albumin, FC, ESR, and the SNP rs1048610 accurately predicted infliximab trough concentrations in children with IBD.

Blood gene expression biomarkers of response to anti-TNF drugs in pediatric inflammatory bowel diseases before initiation of treatment.

BACKGROUND/AIMS: Changes in gene expression profiles among individuals with inflammatory bowel diseases (IBDs) could potentially influence the responsiveness to anti-TNF treatment. The aim of this study was to identify genes that could serve as predictors of early response to anti-TNF therapies in pediatric IBD patients prior to the initiation of treatment. METHODS: We conducted a prospective, longitudinal, and multicenter study, enrolling 24 pediatric IBD patients aged less than 18 years who were initiating treatment with either infliximab or adalimumab. RNA-seq from blood samples was analyzed using the DESeq2 library by comparing responders and non-responders to anti-TNF drugs. RESULTS: Bioinformatic analyses unveiled 102 differentially expressed genes, with 99 genes exhibiting higher expression in responders compared to non-responders prior to the initiation of anti-TNF therapy. Functional enrichment analyses highlighted defense response to Gram-negative bacteria (FDR = 2.3 ×10-7) as the most significant biological processes, and hemoglobin binding (FDR = 0.002), as the most significant molecular function. Gene Set Enrichment Analysis (GSEA) revealed notable enrichment in transcriptional misregulation in cancer (FDR = 0.016). Notably, 13 genes (CEACAM8, CEACAM6, CILP2, COL17A1, OLFM4, INHBA, LCN2, LTF, MMP8, DEFA4, PRTN3, AZU1, and ELANE) were selected for validation, and a consistent trend of increased expression in responders prior to drug administration was observed for most of these genes, with findings for 4 of them being statistically significant (CEACAM8, LCN2, LTF2, and PRTN3). CONCLUSIONS: We identified 102 differentially expressed genes involved in the response to anti-TNF drugs in children with IBDs and validated CEACAM8, LCN2, LTF2, and PRTN3. Genes participating in defense response to Gram-negative bacterium, serine-type endopeptidase activity, and transcriptional misregulation in cancer are good candidates for anticipating the response to anti-TNF drugs in children with IBDs.

Changes in the Expression of TGF-Beta Regulatory Pathway Genes Induced by Vitamin D in Patients with Relapsing-Remitting Multiple Sclerosis.

Vitamin D is an environmental factor related to multiple sclerosis that plays a significant role in immune regulation. TGF-ß is a superfamily of cytokines with an important dual effect on the immune system. TGF-ß inhibits the Th1 response while facilitating the preservation of regulatory T cells (FOXP3+) in an immunoregulatory capacity. However, when IL-6 is present, it stimulates the Th17 response. Our aim was to analyze the regulatory effect of vitamin D on the in vivo TGF-ß signaling pathway in patients with relapsing-remitting multiple sclerosis (RRMS). A total of 21 patients with vitamin D levels < 30 ng/mL were recruited and supplemented with oral vitamin D. All patients were receiving disease-modifying therapy, with the majority being on natalizumab. Expression of SMAD7, ERK1, ZMIZ1, BMP2, BMPRII, BMP4, and BMP5 was measured in CD4+ lymphocytes isolated from peripheral blood at baseline and one and six months after supplementation. SMAD7 was overexpressed at six months with respect to baseline and month one. ERK1 was overexpressed at six months with respect to month one of treatment. No significant differences in expression were observed for the remaining genes. No direct correlation was found with serum vitamin D levels. BMPRII expression changed differentially in non-natalizumab- versus natalizumab-treated patients. Changes were observed in the expression of ERK1, BMP2, and BMP5 based on disease activity measured using the Rio-Score, BMP2 in patients who had relapses, and BMP5 in those whose EDSS worsened. Our results suggest indirect regulation of vitamin D in TGF-ß pathway genes in patients with RRMS.

Polymorphisms indicating risk of inflammatory bowel disease or antigenicity to anti-TNF drugs as biomarkers of response in children.

Few genetic polymorphisms predict early response to anti-TNF drugs in inflammatory bowel disease (IBD), and even fewer have been identified in the pediatric population. However, it would be of considerable clinical interest to identify and validate genetic biomarkers of long-term response. Therefore, the aim of the study was to analyze the usefulness of biomarkers of response to anti-TNFs in pediatric IBD (pIBD) as long-term biomarkers and to find differences by type of IBD and type of anti-TNF drug. The study population comprised 340 children diagnosed with IBD who were treated with infliximab or adalimumab. Genotyping of 9 selected SNPs for their association with early response and/or immunogenicity to anti-TNFs was performed using real-time PCR. Variants C rs10508884 (CXCL12), A rs2241880 (ATG16L1), and T rs6100556 (PHACTR3) (p value 0.049; p value 0.03; p value 0.031) were associated with worse long-term response to anti-TNFs in pIBD. DNA variants specific to disease type and anti-TNF type were identified in the pediatric population. Genotyping of these genetic variants before initiation of anti-TNFs would enable, if validated in a prospective cohort, the identification of pediatric patients who are long-term responders to this therapy.

Association between HLA DNA Variants and Long-Term Response to Anti-TNF Drugs in a Spanish Pediatric Inflammatory Bowel Disease Cohort.

The genetic polymorphisms rs2395185 and rs2097432 in HLA genes have been associated with the response to anti-TNF treatment in inflammatory bowel disease (IBD). The aim was to analyze the association between these variants and the long-term response to anti-TNF drugs in pediatric IBD. We performed an observational, multicenter, ambispective study in which we selected 340 IBD patients under 18 years of age diagnosed with IBD and treated with anti-TNF drugs from a network of Spanish hospitals. Genotypes and failure of anti-TNF drugs were analyzed using Kaplan-Meier curves and Cox logistic regression. The homozygous G allele of rs2395185 and the C allele of rs2097432 were associated with impaired long-term response to anti-TNF drugs in children with IBD after 3 and 9 years of follow-up. Being a carrier of both polymorphisms increased the risk of anti-TNF failure. The SNP rs2395185 but not rs2097432 was associated with response to infliximab in adults with CD treated with infliximab but not in children after 3 or 9 years of follow-up. Conclusions: SNPs rs2395185 and rs2097432 were associated with a long-term response to anti-TNFs in IBD in Spanish children. Differences between adults and children were observed in patients diagnosed with CD and treated with infliximab.

Genotyping of UGT1A1*80 as an Alternative to UGT1A1*28 Genotyping in Spain.

BACKGROUND: The variant rs34983651 (UGT1A1*28) and its genotyping are used to prevent irinotecan-induced toxicity. Several variants are in close linkage disequilibrium. Our objective was to evaluate the potential correlation of genotyping UGT1A1*80 instead of UGT1A1*28 in different populations. METHODS: We studied SNPs in linkage disequilibrium with UGT1A1*28 in several populations and selected rs887829 to develop an inexpensive and rapid genotyping method and compare it with the one we currently use for UGT1A1*28 genotyping. Samples from cancer patients (n = 701) already tested using PCR and electrophoresis prior to treatment with irinotecan for rs34983651 (UGT1A1*28) in a Spanish hospital were genotyped for rs887829 (UGT1A1*80) using real-time PCR with a TaqMan probe. RESULTS: We observed a complete match for both genotypes, except in one sample. This method was 100% efficient in correctly genotyping *28/*28 patients, 99.68% efficient for *1/*28, and 100% efficient for *1/*1. Linkage disequilibrium between populations showed the Iberian population to be the most suitable for the clinical use of UGT1A1*80. This method is less expensive and the time to decision is shorter. CONCLUSION: Genotyping of rs887829 using the proposed method may be used to substitute genotyping of rs34983651 as a pharmacogenetics test in cancer patients prior to starting irinotecan-based treatments, mainly in the Iberian population. In addition, it is less expensive than other conventional methods and easy to implement, with a shorter time to decision than UGT1A1*28.

Pharmacogenetics to Avoid Adverse Drug Reactions.

Although a cure is the main goal of a treatment, serious adverse reactions associated with these treatments are a major problem in clinical practice and cost a lot of money for health systems [...].

ABO blood group is involved in the quality of the specific immune response anti-SARS-CoV-2.

Since December 2019, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. To eradicate it, it is crucial to acquire a strong and long-lasting anti-SARS-CoV-2 immunity, by either natural infection or vaccination. We collected blood samples 12-305 days after positive polymerase chain reactions (PCRs) from 35 recovered individuals infected by SARS-CoV-2. Peripheral blood mononuclear cells were stimulated with SARS-CoV-2-derived peptide pools, such as the spike (S), nucleocapsid (N) and membrane (M) proteins, and we quantified anti-S immunoglobulins in plasma. After 10 months post-infection, we observed a sustained SARS-CoV-2-specific CD4+ T-cell response directed against M-protein, but responses against S- or N-proteins were lost over time. Besides, we demonstrated that O-group individuals presented significantly lower frequencies of specific CD4+ T-cell responses against Pep-M than non O-group individuals. The non O-group subjects also needed longer to clear the virus, and they lost cellular immune responses over time, compared to the O-group individuals, who showed a persistent specific immune response against SARS-CoV-2. Therefore, the S-specific immune response was lost over time, and individual factors might determine the sustainability of the body's defenses, which must be considered in the future design of vaccines to achieve continuous anti-SARS-CoV-2 immunity.

Clinical Relevance of Novel Polymorphisms in the Dihydropyrimidine Dehydrogenase (DPYD) Gene in Patients with Severe Fluoropyrimidine Toxicity: A Spanish Case-Control Study.

Among cancer patients treated with fluoropyrimidines, 10-40% develop severe toxicity. Polymorphism of the dihydropyrimidine dehydrogenase (DPYD) gene may reduce DPD function, the main enzyme responsible for the metabolism of fluoropyrimidines. This leads to drug accumulation and to an increased risk of toxicity. Routine genotyping of this gene, which usually includes DPYD *HapB3, *2A, *13 and c.2846A > T (D949V) variants, helps predict approximately 20-30% of toxicity cases. For DPD intermediate (IM) or poor (PM) metabolizers, a dose adjustment or drug switch is warranted to avoid toxicity, respectively. Societies such as the Spanish Society of Pharmacogenetics and Pharmacogenomics (SEFF), the Dutch Pharmacogenetics Working Group (DPWG) or the Clinical Pharmacogenetics Implementation Consortium (CPIC) and regulatory agencies (e.g., the Spanish Medicines Agency, AEMPS) already recommend DPYD routine genotyping. However, the predictive capacity of genotyping is currently still limited. This can be explained by the presence of unknown polymorphisms affecting the function of the enzyme. In this case-control work, 11 cases of severe fluoropyrimidine toxicity in patients who did not carry any of the four variants mentioned above were matched with 22 controls, who did not develop toxicity and did not carry any variant. The DPYD exome was sequenced (Sanger) in search of potentially pathogenic mutations. DPYD rs367619008 (c.187 A > G, p.Lys63Glu), rs200643089 (c.2324 T > G, p.Leu775Trp) and rs76387818 (c.1084G > A, p.Val362Ile) increased the percentage of explained toxicities to 38-48%. Moreover, there was an intronic variant considered potentially pathogenic: rs944174134 (c.322-63G > A). Further studies are needed to confirm its clinical relevance. The remaining variants were considered non-pathogenic.

Biomarkers for Optimization and Personalization of Anti-TNFs in Pediatric Inflammatory Bowel Disease.

The use of biological drugs has improved outcomes in pediatric inflammatory bowel disease (IBD). Prediction of the response to biological drugs would be extremely useful in IBD, and even more so in children, who are still growing physically and psychologically. Specific clinical, biochemical, and genetic parameters are considered predictive of response to biological drugs, although few studies have been carried out in children with IBD. In this review, we present current evidence on biological treatments used in pediatric IBD and the available biomarkers of response. We examine demographics, clinical characteristics, biomarkers (genetic, genomic, and cellular), and microbiota.

Induction of High Levels of Specific Humoral and Cellular Responses to SARS-CoV-2 After the Administration of Covid-19 mRNA Vaccines Requires Several Days.

Objectives: In the context of the Covid-19 pandemic, the fast development of vaccines with efficacy of around 95% preventing Covid-19 illness provides a unique opportunity to reduce the mortality associated with the pandemic. However, in the absence of efficacious prophylactic medications and few treatments for this infection, the induction of a fast and robust protective immunity is required for effective disease control, not only to prevent the disease but also the infection and shedding/transmission. The objective of our study was to analyze the level of specific humoral and cellular T-cell responses against the spike protein of SARS-CoV-2 induced by two mRNA-based vaccines (BNT162b2 and mRNA-1273), but also how long it takes after vaccination to induce these protective humoral and cellular immune responses. Methods: We studied in 40 healthy (not previously infected) volunteers vaccinated with BNT162b2 or mRNA-1273 vaccines the presence of spike-specific IgG antibodies and SARS-CoV-2-specific T cells at 3, 7 and 14 days after receiving the second dose of the vaccine. The specific T-cell response was analyzed stimulating fresh whole blood from vaccinated volunteers with SARS-CoV-2 peptides and measuring the release of cytokines secreted by T cells in response to SARS-CoV-2 stimulation. Results: Our results indicate that the immunization capacity of both vaccines is comparable. However, although both BNT162b2 and mRNA-1273 vaccines can induce early B-cell and T-cell responses, these vaccine-mediated immune responses do not reach their maximum values until 14 days after completing the vaccination schedule. Conclusion: This refractory period in the induction of specific immunity observed after completing the vaccination could constitute a window of higher infection risk, which could explain some emerging cases of SARS-CoV-2 infection in vaccinated people.

DPYD Exome, mRNA Expression and Uracil Levels in Early Severe Toxicity to Fluoropyrimidines: An Extreme Phenotype Approach.

Dihydropyrimidine dehydrogenase deficiency is a major cause of severe fluoropyrimidine-induced toxicity and could lead to interruption of chemotherapy or life-threatening adverse reactions. This study aimed to characterize the DPYD exon sequence, mRNA expression and in vivo DPD activity by plasma uracil concentration. It was carried out in two groups of patients with extreme phenotypes (toxicity versus control) newly treated with a fluoropyrimidine, during the first three cycles of treatment. A novel nonsense gene variant (c.2197insA) was most likely responsible for fluoropyrimidine-induced toxicity in one patient, while neither DPYD mRNA expression nor plasma uracil concentration was globally associated with early toxicity. Our present work may help improve pharmacogenetic testing to avoid severe and undesirable adverse reactions to fluoropyrimidine treatment and it also supports the idea of looking beyond DPYD.

Comparison of a new rapid method for determination of serum anti-adalimumab and anti-infliximab antibodies with two established ELISA kits.

BACKGROUND: Adalimumab (ADL), infliximab (IFX) and their biosimilars are widely used biological drugs. Some patients, however, generate neutralizing antibodies that hamper the effectiveness of these drugs. Evidence shows therapeutic drug monitoring of serum levels ADL/IFX and anti-drug antibodies (ADA) is useful to improve treatment effectiveness. We evaluated a new rapid quantitative method, Quantum Blue (QB), for determining serum anti-ADL and anti-IFX antibodies (Research Use Only labelling) by comparing it with two established ELISA kits, Promonitor (PM) and Lisa-Tracker (LT). METHODS: Eighty samples (40 for each drug type) were analysed. Percentage of agreement and kappa statistic were used to compare positive/negative ADA results. Clinical implications for drug treatment in the patients with discordant results were evaluated. The Chi-square test was used to analyze differences for ADA detection in patients with disease flare and without concomitant immunosuppressant treatment. RESULTS: Agreement exceeded 80 % among anti-ADL methods. Although LT ELISA showed a lower capacity in detecting anti-ADL antibodies, discrepancies were found for levels close to the cut-off concentration, thus having minimal impact on clinical decisions. Conversely, QB anti-IFX displayed low agreement with PM and LT ELISA kits (67.5 % and 50 %, respectively), and was unable to detect high levels of antibodies, therefore having major clinical implications. Agreement between PM and LT ELISA anti-IFX kits was 82.5 % with all discordant results being undetected for PM and slightly positive for LT. CONCLUSION: QB anti-ADL shows similar performance to ELISA kits while QB anti-IFX needs further improvements to achieve reliable antibody detection.

Whole Transcription Profile of Responders to Anti-TNF Drugs in Pediatric Inflammatory Bowel Disease.

BACKGROUND: Up to 30% of patients with pediatric inflammatory bowel disease (IBD) do not respond to anti-Tumor Necrosis Factor (anti-TNF) therapy. The aim of this study was to identify pharmacogenomic markers that predict early response to anti-TNF drugs in pediatric patients with IBD. METHODS: An observational, longitudinal, prospective cohort study was conducted. The study population comprised 38 patients with IBD aged < 18 years who started treatment with infliximab or adalimumab (29 responders and nine non-responders). Whole gene expression profiles from total RNA isolated from whole blood samples of six responders and six non-responders taken before administration of the biologic and after two weeks of therapy were analyzed using next-generation RNA sequencing. The expression of six selected genes was measured for purposes of validation in all of the 38 patients recruited using qPCR. RESULTS: Genes were differentially expressed in non-responders and responders (32 before initiation of treatment and 44 after two weeks, Log2FC (Fold change) >0.6 or <-0.6 and p value < 0.05). After validation, FCGR1A, FCGR1B, and GBP1 were overexpressed in non-responders two weeks after initiation of anti-TNF treatment (Log2FC 1.05, 1.21, and 1.08, respectively, p value < 0.05). CONCLUSION: Expression of the FCGR1A, FCGR1B, and GBP1 genes is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD.

Comprehensive Flow Cytometry Profiling of the Immune System in COVID-19 Convalescent Individuals.

SARS-CoV-2 has infected more than 200 million people worldwide, with more than 4 million associated deaths. Although more than 80% of infected people develop asymptomatic or mild COVID-19, SARS-CoV-2 can induce a profound dysregulation of the immune system. Therefore, it is important to investigate whether clinically recovered individuals present immune sequelae. The potential presence of a long-term dysregulation of the immune system could constitute a risk factor for re-infection and the development of other pathologies. Here, we performed a deep analysis of the immune system in 35 COVID-19 recovered individuals previously infected with SARS-CoV-2 compared to 16 healthy donors, by flow cytometry. Samples from COVID-19 individuals were analysed from 12 days to 305 days post-infection. We observed that, 10 months post-infection, recovered COVID-19 patients presented alterations in the values of some T-cell, B-cell, and innate cell subsets compared to healthy controls. Moreover, we found in recovered COVID-19 individuals increased levels of circulating follicular helper type 1 (cTfh1), plasmablast/plasma cells, and follicular dendritic cells (foDC), which could indicate that the Tfh-B-foDC axis might be functional to produce specific immunoglobulins 10 months post-infection. The presence of this axis and the immune system alterations could constitute prognosis markers and could play an important role in potential re-infection or the presence of long-term symptoms in some individuals.

Regulatory CDH4 Genetic Variants Associate With Risk to Develop Capecitabine-Induced Hand-Foot Syndrome.

Capecitabine-induced hand-foot syndrome (CiHFS) is a common dermatological adverse reaction affecting around 30% of patients with capecitabine-treated cancer, and the main cause of dose reductions and chemotherapy delays. To identify novel genetic factors associated with CiHFS in patients with cancer, we carried out an extreme-phenotype genomewide association study in 166 patients with breast and colorectal capecitabine-treated cancer with replication in a second cohort of 85 patients. We discovered and replicated a cluster of four highly correlated single-nucleotide polymorphisms associated with susceptibility to CiHFS at 20q13.33 locus (top hit = rs6129058, hazard ratio = 2.40, 95% confidence interval = 1.78-3.20; P = 1.2 × 10-8 ). Using circular chromosome conformation capture sequencing, we identified a chromatin contact between the locus containing the risk alleles and the promoter of CDH4, located 90 kilobases away. The risk haplotype was associated with decreased levels of CDH4 mRNA and the protein it encodes, R-cadherin (RCAD), which mainly localizes in the granular layer of the epidermis. In human keratinocytes, CDH4 downregulation resulted in reduced expression of involucrin, a protein of the cornified envelope, an essential structure for skin barrier function. Immunohistochemical analyses revealed that skin from patients with severe CiHFS exhibited low levels of RCAD and involucrin before capecitabine treatment. Our results uncover a novel mechanism underlying individual genetic susceptibility to CiHFS with implications for clinically relevant risk prediction.