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Background Human milk (HM) is the first form of communication between mothers and newborns and it is implicated in the infant growth and protection. We recently showed a functional characterization of HM, unmasking the molecular mechanisms related to EVs signaling and its functional role in prematurity. In that study, we identified the need to establish and optimize a standard isolation protocol for human milk extracellular vesicles (mEVs). Methods Four mEVs isolation methods were compared: ultracentrifugation (UC), size exclusion chromatography (SEC), immunoprecipitation with tetraspanin CD9 (IP_CD9) and ExoGAG. Three pools of human milk (each composed of samples from ten donor mothers) were used and isolation of mEVs was performed starting from the same volume for each method. The proteomic, transcriptomic and glycomic composition of the extracellular vesicles obtained after isolation was then analyzed for each method. The sensitivity, specificity and quality of the results were also determined. A comparative analysis of results common across all isolation methods was performed to identify potential signaling pathways associated with mEVs. Results ExoGAG and UC proved to be the most efficient of the four techniques compared for mEVs isolation. However, ExoGAG compared to UC provided a higher concentration of total and vesicle-related proteins and peptides and a higher glycoprotein count keeping all the glycan subgroups. Despite ExoGAG and UC show similar vesicle profiles in terms of size, concentration, tetraspanin subpopulations and EVs markers, ExoGAG was the most efficient technique in terms of accuracy, consistency and reproducibility for omics studies. Furthermore, results allowed us to identify that mEVs components are involved in the signaling pathways of infant biological development, immune system maturation and protein metabolism. Conclusions This study establishes UC and ExoGAG as reliable methods for mEVs isolation and describes its protocol, being ExoGAG the most efficient. Also, the omics analysis show that biomolecules conforming those mEVs are linked to the defense system against external agents (specific role in the immune system pathway) and in the correct establishment of the neural structure (developmental pathway), while providing all the nutritional requirements for the correct growth of the newborn (metabolic pathway). Graphical Abstract

The pediatric population is a vulnerable group for adverse drug reactions (ADRs), and data on spontaneous reporting of ADRs in the hospital setting are scarce. We conducted a retrospective analysis of ADRs in pediatric patients spontaneously reported by health care professionals to a Pharmacovigilance Program in a tertiary hospital between 2010 and 2020, and we compared characteristics of ADRs between pediatric age subgroups. From 1787 spontaneously reported ADRs in an 11-year period, 103 (5.85%) were pediatric ADRs. The median age of patients with ADRs was 8.4 years (range 1 day–17 years) and 57.3% were male. The most frequent ADRs reported were nervous system disorders (13.6%) and the most frequently involved drugs were antineoplastics and immunodulators (32.4%). A 59.2% of the ADRs were serious and 55.3% were classified as being type B reactions. Medication errors were involved in 7.8% of the ADRs and 11.9% of the suspected drugs were used off-label. Spontaneous reports of ADRs in newborns, infants, and toddlers were more serious and less often described in the product data sheet than in children and adolescents (p < 0.001 and p = 0.004 respectively). Medication errors were more frequent in patients under two years of age. These results should be interpreted with caution due to under-reporting and biases in spontaneous reporting of ADRs.

Mucolipidosis type II is a very rare lysosomal disease affecting the UDP-GlcNAc N-acetylglucosamine-1-phosphotransferase enzyme, which catalyzes the synthesis of the targeting signal mannose 6-phosphate in lysosomal acid hydrolases. Its deficiency hinders the arrival of lysosomal enzymes to the lysosome, diminishing the multiple degradations of components that cells need to perform. Due to the low prevalence of this condition, available information is scarce. This article aims to deepen the understanding of the disease; clinical, biochemical, and proteomic data are analyzed. Three patients have been identified presenting GNPTAB pathogenic variants using whole exome sequencing. A biochemical profile for these patients has been carried out through quantification of glycosaminoglycans in urine samples and enzymatic analysis in dried blood spot (DBS) samples. Quantitative proteomic studies were performed. Results show how enzymatic assays in DBS can be used to diagnose this disease both during the neonatal period or in patients of more advanced age. Increased levels of acid sphingomyelinase, alpha-iduronidase, iduronidate 2-sulfatase, alpha-N-acetyl glucosaminidase, and beta-glucuronidase are found. Conclusion: this biochemical method could potentially improve early diagnosis. Proteomic data supporting these results reveal disrupted biochemical pathways, including the degradation of dermatan sulfate, heparan sulfate, and cellular cholesterol trafficking.

Pompe disease (PD) is a rare, autosomal recessive neuromuscular disorder caused by mutations in the gene encoding acid alpha-glucosidase ( ). The resulting deficiency in GAA, a lysosomal enzyme, leads to the pathological accumulation of glycogen, primarily in cardiac and skeletal muscles. PD presents as a clinical continuum spanning two major phenotypes: infantile-onset Pompe disease (IOPD), the most severe form, typically characterized by onset before 12 months of age, rapid hypertrophic cardiomyopathy, and severe hypotonia; and late-onset Pompe disease (LOPD), which manifests between 12 months of age and adulthood, and is characterized by progressive axial and proximal muscle weakness and respiratory insufficiency. Enzyme replacement therapy (ERT), available since 2006, has improved survival, particularly in IOPD, but is limited by variable efficacy and limited penetration of the blood-brain barrier, necessitating new approaches. In this comprehensive review, we focus on advances in the understanding and management of PD. First, we explore recent diagnostic advances and the characterization of multisystem involvement in PD. Next, we critically discuss the advantages and limitations of current ERT approaches, and advances achieved with next-generation ERT (avalglucosidase alfa, cipaglucosidase alfa + miglustat). Finally, we summarize cutting-edge, potentially curative strategies, including substrate reduction therapy and novel experimental therapies (e.g., gene therapy) that seek to circumvent the limitations of ERT, provide durable effects, and potentially penetrate the central nervous system.

People living with HIV have accounted for 38–50% of those affected in the 2022 multicountry mpox outbreak. Most reported cases were in people who had high CD4 cell counts and similar outcomes to those without HIV. Emerging data suggest worse clinical outcomes and higher mortality in people with more advanced HIV. We describe the clinical characteristics and outcomes of mpox in a cohort of people with HIV and low CD4 cell counts (CD4 <350 cells per mm3). A network of clinicians from 19 countries provided data of confirmed mpox cases between May 11, 2022, and Jan 18, 2023, in people with HIV infection. Contributing centres completed deidentified structured case report sheets to include variables of interest relevant to people living with HIV and to capture more severe outcomes. We restricted this series to include only adults older than 18 years living with HIV and with a CD4 cell count of less than 350 cells per mm3 or, in settings where a CD4 count was not always routinely available, an HIV infection clinically classified as US Centers for Disease Control and Prevention stage C. We describe their clinical presentation, complications, and causes of death. Analyses were descriptive. We included data of 382 cases: 367 cisgender men, four cisgender women, and ten transgender women. The median age of individuals included was 35 (IQR 30–43) years. At mpox diagnosis, 349 (91%) individuals were known to be living with HIV; 228 (65%) of 349 adherent to antiretroviral therapy (ART); 32 (8%) of 382 had a concurrent opportunistic illness. The median CD4 cell count was 211 (IQR 117–291) cells per mm3, with 85 (22%) individuals with CD4 cell counts of less than 100 cells per mm3 and 94 (25%) with 100–200 cells per mm3. Overall, 193 (51%) of 382 had undetectable viral load. Severe complications were more common in people with a CD4 cell count of less than 100 cells per mm3 than in those with more than 300 cells per mm3, including necrotising skin lesions (54% vs 7%), lung involvement (29% vs 0%) occasionally with nodules, and secondary infections and sepsis (44% vs 9%). Overall, 107 (28%) of 382 were hospitalised, of whom 27 (25%) died. All deaths occurred in people with CD4 counts of less than 200 cells per mm3. Among people with CD4 counts of less than 200 cells per mm3, more deaths occurred in those with high HIV viral load. An immune reconstitution inflammatory syndrome to mpox was suspected in 21 (25%) of 85 people initiated or re-initiated on ART, of whom 12 (57%) of 21 died. 62 (16%) of 382 received tecovirimat and seven (2%) received cidofovir or brincidofovir. Three individuals had laboratory confirmation of tecovirimat resistance. A severe necrotising form of mpox in the context of advanced immunosuppression appears to behave like an AIDS-defining condition, with a high prevalence of fulminant dermatological and systemic manifestations and death. None.