Breast milk is an essential source of nutrition and hydration for the infant. In addition, this highly complex fluid is rich in extracellular vesicles (EVs). Here, we have applied a microfluidic technology, lipid-based protein immobilization (LPI) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) to characterize the proteome of human milk EVs. Mature milk from six mothers was subjected to EV isolation by ultracentrifugation followed by size exclusion chromatography. Three of the samples were carefully characterized; suggesting a subset enriched by small EVs. The EVs were digested by trypsin in an LPI flow cell and in-solution digestion, giving rise to two fractions of peptides originating from the surface proteome (LPI fraction) or the complete proteome (in-solution digestion). LC-MS/MS recovered peptides corresponding to 582 proteins in the LPI fraction and 938 proteins in the in-solution digested samples; 400 of these proteins were uniquely found in the in-solution digested samples and were hence denoted "cargo proteome". GeneOntology overrepresentation analysis gave rise to distinctly different functional predictions of the EV surfaceome and the cargo proteome. The surfaceome tends to be overrepresented in functions and components of relevance for the immune system, while the cargo proteome primarily seems to be associated with EV biogenesis.

Background: Tinzaparin, a low-molecular weight heparin (LMWH), has shown anti-neoplastic properties in animal models and in in vitro studies of human cancer cell lines. The reduction of CA-125 levels during neoadjuvant chemotherapy (NACT) in patients with epithelial ovarian cancer (EOC) co-varies with the prognosis; the larger the decrease in CA-125, the better the prognosis.

Purpose: This study aims to evaluate the potential anti-neoplastic effects of tinzaparin by investigating changes in serum CA-125 levels in advanced EOC patients who receive NACT.

Material and methods: This is an open randomized multicenter pilot trial. Forty patients with EOC selected to receive NACT will be randomized 1:1 to receive daily addition of tinzaparin or no tinzaparin. The processing and treatment of the patients will otherwise follow the recommendations in the Swedish National Guidelines for Ovarian Cancer. Before every cycle of chemotherapy, preoperatively, and 3 weeks after the last cycle of chemotherapy, a panel of biomarkers, including CA-125, will be measured.

Patients: Inclusion criteria are women aged 18 years or older, World Health Organization performance status 0–1, histologically confirmed high-grade serous, endometrioid or clear cell EOC, International Federation of Gynecology and Obstetrics (FIGO) stages III-IV. In addition, a CA-125 level of ≥ 250 kIE/L at diagnosis. Exclusion criteria are contraindications to LMWH, ongoing or recent treatment with unfractionated heparin, LMWH, warfarin or non-vitamin K antagonist oral anticoagulants.

Interpretation: This study will make an important contribution to the knowledge of the anti-neoplastic effects of tinzaparin in EOC patients and may thus guide the planning of a future study on the impact of tinzaparin on survival in EOC. 

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (β-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein–Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination. Copyright © 2023 the Author(s).

Sensitive and reliable protein biomarkers are needed to predict disease trajectory and personalize treatment strategies for multiple sclerosis (MS). Here, we use the highly sensitive proximity-extension assay combined with next-generation sequencing (Olink Explore) to quantify 1463 proteins in cerebrospinal fluid (CSF) and plasma from 143 people with early-stage MS and 43 healthy controls. With longitudinally followed discovery and replication cohorts, we identify CSF proteins that consistently predicted both short- and long-term disease progression. Lower levels of neurofilament light chain (NfL) in CSF is superior in predicting the absence of disease activity two years after sampling (replication AUC = 0.77) compared to all other tested proteins. Importantly, we also identify a combination of 11 CSF proteins (CXCL13, LTA, FCN2, ICAM3, LY9, SLAMF7, TYMP, CHI3L1, FYB1, TNFRSF1B and NfL) that predict the severity of disability worsening according to the normalized age-related MS severity score (replication AUC = 0.90). The identification of these proteins may help elucidate pathogenetic processes and might aid decisions on treatment strategies for persons with MS.

Introduction

There is a need for a fast, efficient and safe way to induce tolerance in patients with severe allergic rhinitis. Intralymphatic immune therapy has been shown to be effective.

Methods

Patients with severe birch and timothy allergy were randomized and received three doses of 0.1 ml of birch and 5-grass allergen extracts (10,000 SQ units/ml, ALK-Abello), or birch and placebo or 5-grass and placebo by ultrasound-guided injections into inguinal lymph nodes at monthly intervals. Rhinoconjunctivitis total symptom score, medication score and rhinoconjunctivitis quality of life questionnaire were evaluated before treatment and after each birch and grass pollen season during three subsequent years. Circulating proportions of T helper subsets and allergen-induced cytokine and chemokine production were analysed by flow cytometry and Luminex.

Results

The three groups reported fewer symptoms, lower use of medication and improved quality of life during the birch and grass pollen seasons each year after treatment at an almost similar rate independently of treatment with one or two allergens. Mild local pain was the most common adverse event. IgE levels to birch decreased, whereas birch-induced IL-10 secretion increased in all three groups. IgG4 levels to birch and timothy and skin prick test reactivity remained mainly unchanged. Conjunctival challenge tests with timothy extract showed a higher threshold for allergen. In all three groups, regulatory T cell frequencies were increased 3 years after treatment.

Conclusions

Intralymphatic immunotherapy with one or two allergens in patients with grass and birch pollen allergy was safe, effective and may be associated with bystander immune modulatory responses.

16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic L. reuteri identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiotic supplementation and clinical outcomes. It is customizable for other clinical trials. For complete details on the use and execution of this protocol, please refer to Martí et al. (2021) and Spreckels et al. (2021).

BackgroundEnvironmental exposures may alter DNA methylation patterns of T helper cells. As T helper cells are instrumental for allergy development, changes in methylation patterns may constitute a mechanism of action for allergy preventive interventions. While epigenetic effects of separate perinatal probiotic or omega -3 fatty acid supplementation have been studied previously, the combined treatment has not been assessed. We aimed to investigate epigenome-wide DNA methylation patterns from a sub-group of children in an on-going randomised double-blind placebo-controlled allergy prevention trial using pre- and postnatal combined Lactobacillus reuteri and omega -3 fatty acid treatment. To this end,>866000 CpG sites (MethylationEPIC 850K array) in cord blood CD4+ T cells were examined in samples from all four study arms (double-treatment: n=18, single treatments: probiotics n=16, omega -3 n=15, and double placebo: n=14). Statistical and bioinformatic analyses identified treatment-associated differentially methylated CpGs and genes, which were used to identify putatively treatment-induced network modules. Pathway analyses inferred biological relevance, and comparisons were made to an independent allergy data set.ResultsComparing the active treatments to the double placebo group, most differentially methylated CpGs and genes were hypermethylated, possibly suggesting induction of transcriptional inhibition. The double-treated group showed the largest number of differentially methylated CpGs, of which many were unique, suggesting synergy between interventions. Clusters within the double-treated network module consisted of immune-related pathways, including T cell receptor signalling, and antigen processing and presentation, with similar pathways revealed for the single-treatment modules. CpGs derived from differential methylation and network module analyses were enriched in an independent allergy data set, particularly in the double-treatment group, proposing treatment-induced DNA methylation changes as relevant for allergy development.ConclusionPrenatal L. reuteri and/or omega -3 fatty acid treatment results in hypermethylation and affects immune- and allergy-related pathways in neonatal T helper cells, with potentially synergistic effects between the interventions and relevance for allergic disease. Further studies need to address these findings on a transcriptional level, and whether the results associate to allergy development in the children. Understanding the role of DNA methylation in regulating effects of perinatal probiotic and omega -3 interventions may provide essential knowledge in the development of efficacious allergy preventive strategies.Trial registration ClinicalTrials.gov, ClinicalTrials.gov-ID: NCT01542970. Registered 27th of February 2012-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01542970.

Introduction

Preterm labor is a common clinical problem in obstetrics. Since the majority of women with preterm labor eventually deliver at full term, biomarkers are needed to more accurately predict who will deliver preterm. Oxylipins, given their importance in inflammation regulation, are highly interesting in this respect since labor is an inflammatory process.

Methods

Eighty women with preterm labor before 34 weeks of gestation were enrolled in a prospective observational multi-center cohort study. Oxylipin levels of 67 analytes in plasma samples were analyzed by liquid chromatography coupled to tandem mass spectrometry.

Results

Twenty-one (26%) of the women delivered before 34 weeks of gestation, and of those women, fourteen delivered within 48 h of admission. Logistic multivariate regression showed that lower levels of 9,10-DiHODE were associated with delivery before 34 weeks of gestation (aOR 0.12 (0.024–0.62)) and within 48 h ((aOR 0.13 (0.019–0.93)). Furthermore, higher levels of 11,12-DiHETrE were associated with delivery before 34 weeks of gestation ((aOR 6.19 (1.17–32.7)) and higher levels of 8-HETE were associated with delivery within 48 h ((aOR 5.01 (1.13–22.14)).

Conclusions

The oxylipin 9,10-DiHODE may be protective in preterm labor, both for delivery after 34 weeks of gestation and for delivery later than 48 h of admission, whereas 11,12-DiHETrE and 8-HETE display the opposite effect. Larger studies are needed to validate these mediators as biomarkers for prediction of preterm birth following preterm labor.

Background Early colonization with a diverse microbiota seems to play a crucial role for appropriate immune maturation during childhood. Breastmilk microbiota is one important source of microbes for the infant, transferred together with maternal IgA antibodies. We previously observed that allergy development during childhood was associated with aberrant IgA responses to the gut microbiota already at 1 month of age, when the IgA antibodies are predominantly maternally derived in breastfed infants. Objective To determine the microbial composition and IgA-coated bacteria in breastmilk in relation to allergy development in children participating in an intervention trial with pre- and post-natal Lactobacillus reuteri supplementation. Methods A combination of flow cytometric cell sorting and 16S rRNA gene sequencing was used to characterize the bacterial recognition patterns by IgA in breastmilk samples collected one month post-partum from 40 mothers whose children did or did not develop allergic and asthmatic symptoms during the first 7 years of age. Results The milk fed to children developing allergic manifestations had significantly lower bacterial richness, when compared to the milk given to children that remained healthy. Probiotic treatment influenced the breastmilk microbiota composition. However, the proportions of IgA-coated bacteria, the total bacterial load and the patterns of IgA-coating were similar in breastmilk between mothers of healthy children and those developing allergies. Conclusion Consumption of breastmilk with a reduced microbial richness in the first month of life may play an important role in allergy development during childhood.