Commentary: Raw Cow Milk Consumption and Atopic March

We have appreciated the interest of Dr Baars et al. in our paper describing dietary prevention of atopic march (AM) in children affected by cow milk allergy (CMA) (1). They claimed a lack of information on raw cow milk (unpasteurized cow milk) in our paper. In support of this point, they mentioned the result of a pilot study involving nine CMA children (2) that were able to tolerate up to 50 mL of raw milk (about 1,750 mg of cow milk proteins). This result was not confirmed by a similar study where five children with IgE-mediated CMA were orally challenged in a double-blind fashion with raw untreated cow milk, pasteurized cow milk, and homogenized/pasteurized cow milk. An extensively hydrolysed casein formula served as placebo. All patients presented significant allergic reactions from the consumption of the above three types of milk, whereas no adverse reactions to placebo were observed. The authors concluded that children with CMA cannot tolerate raw or pasteurized milk (3). Although, selected components of raw milk may potentially influence the immune system, proof based on controlled studies in children are still lacking (4). The authors of the PARSIFAL study concluded that raw cow milk may contain numerous disease-causing pathogens and that consumption of raw milk cannot be recommended as a preventive measure for allergy (5). Accordingly, none of the claims made by the raw milk advocates (including the postulated preventive effect against allergy) withstand the FDA scientific scrutiny (6)

531. Computational Pipeline for the Identification of Integration Sites and Novel Method for the Quantification of Clone Sizes in Clonal Tracking Studies

Gene-corrected cells in Gene Therapy (GT) treated patients can be tracked in vivo by means of vector integration site (IS) analysis, since each engineered clone becomes univocally and stably marked by an individual IS. As the proper IS identification and quantification is crucial to accurately perform clonal tracking studies, we designed a customizable and tailored pipeline to analyze LAM-PCR amplicons sequenced by Illumina MiSeq/HiSeq technology. The sequencing data are initially processed through a series of quality filters and cleaned from vector and Linker Cassette (LC) sequences with customizable settings. Demultiplexing is then performed according to the recognition of specific barcodes combination used upon library preparation and the sequences are aligned to the reference genome. Importantly, the human genome assembly Hg19 is composed of 93 contigs, among which the mitochondrial genome, unlocalized and unplaced contigs and some alternative haplotypes of chr6. While previous approaches aligned IS sequences only to the standard 24 human chromosomes, using the whole assembled genome allowed improving alignment accuracy and concomitantly increased the amount of detectable ISs. To date, we have processed 28 independent human sample sets retrieving 260,994 ISs from 189,270,566 sequencing reads. Although, sequencing read counts at each IS have been widely used to estimate the relative IS abundance, this method carries inherent accuracy constraints due to the rounds of exponential amplification required by LAM-PCR that might generate unbalances on the original clonal representation. More recently, a method based on genomic sonication has been proposed exploiting shear site counts to tag the number of original fragments belonging to each IS before PCR amplification. However, the number of cells composing a given clone could far exceed the number of fragments of different lengths that can be generated upon fragmentation in proximity of that given IS. This would rapidly saturate the available diversity of shear sites and progressively generate more and more same-site shearing on independent genomes. In order to overcome the described biases and reliably quantify ISs, we designed and tested a new LC encoding random barcodes. The new LC is composed of a known sequence of 29nt used as binding site for the primers upon amplification steps, a 6nt-random barcode, a fixed-anchor sequence of 6nt, a second 6nt-random barcode and a final known sequence of 22nt containing sticky ends for the three main restriction enzymes in use (MluI, HpyCH4IV and AciI). This peculiar design allowed increasing the accuracy of clonal diversity estimation since the fixed-anchor sequence acts as a control for sequencing reliability in the barcode area. The theoretical number of different available barcodes per clone (412=16,777,216) far exceeds the requirements for not saturating the original diversity of the analyzed sample (on average composed by around 50.000 cells). We validated this novel approach by performing assays on serial dilutions of individual clones carrying known ISs. The precision rate obtained was averagely around 99.3%, while the worst error rate reaches at most the 1.86%, confirming the reliability of IS quantification. We successfully applied the barcoded-LC system to the analysis of clinical samples from a Wiskott Aldrich Syndrome GT patient, collecting to date 50,215 barcoded ISs from 94,052,785 sequencing reads

Removal of Koos IV acoustic neuroma and auditory brainstem implant in NF2 patient

The authors present the case of removal of a Koos grade IV right acoustic neuroma in a neurofibromatosis type 2 (NF2) patient, already operated on for left cerebellopontine angle meningioma at 7 years of age and a left acoustic neuroma at 16 years of age. A transpetrosal approach allowed cochlear sensor implantation to detect residual hearing. An enlarged retrosigmoid approach then allowed subtotal microsurgical removal of the lesion; consequently, the authors illustrate the technical nuances of an auditory brainstem implant (ABI). One month after surgery, the ABI was successfully switched on, giving back hearing perception to the patient. The video can be found here: https://stream.cadmore.media/r10.3171/2021.7.FOCVID218

Tolerability of a new amino acid-based formula for children with IgE-mediated cow's milk allergy

Amino acid-based formula (AAF) is a relevant dietary strategy for paediatric patients affected by cow's milk allergy (CMA). The present study was designed to evaluate the hypoallergenicity of a new AAF in children with immunoglobulin (Ig)E-mediated CMA

Tolerogenic effect elicited by protein fraction derived from different hypoallergic formulas in PBMCs from children with cow milk allergy

are available for the dietary treatment of cow’s milk allergy (CMA). Safety and nutritional profile of these formulas have been well evaluated, but the potential tolerogenic activity elicited by their protein fraction is still largely undefined. We aimed to comparatively evaluate the tolerogenic effect elicited by protein fraction derived from different hypoallergenic formulas available for the dietary treatment of CMA METHODS: Four hypoallergenic formulas were compared: extensively whey formula (EHWF), extensively hydrolyzed casein formula (EHCF), hydrolyzed rice formula (RHF), amino acid based formula (AAF). Formulas were reconstituted in water according to manufacturer’s instructions, and subjected to in vitro infant gut simulated digestion using a sequential gastric and duodenal static model. Resulting protein fractions were purified using C18 reversed phase pre-packed cartridges (Sep-Pak, Waters, Milford, MA, USA),recovered in 70% acetonitrile/0.1% trifluoroacetic acid and finally vacuum-dried. Tolerogenic effects were was evaluated in peripheral blood mononuclear cells (PBMCs) from 6 patients, with challenge-proven IgE-mediated CMA (age range 1-5 yrs, all Caucasians), stimulated with different doses of digested protein fractions (from 0.25 to 250 μg/ml) or -lactoglobulin (BLG;100μg/ml) or bovine serum albumin (BSA;100μg/ml) as positive and negative control respectively. The production of Th2 (IL-4, IL-5, IL-13) and Th1 (IL-10, IFN-γ) cytokines were assessed by ELISA. Modulatory action was also evaluated on immune (IL-33) and non-immune tolerogenic factors (mucin 5AC, tight-junction proteins ZO-1 and occludin) in human enterocytes (Caco-2 cells) by ELISA and Real Time PCR, respectively. RESULTS: Th2 cytokines were unaffected by the exposure to protein fraction from all study formulas, whereas only protein fraction from EHCF was able to positively modulate IL-10, IL-33, mucin 5AC, ZO-1 and occludin expression. All protein fraction from study formulas were able to increase INF-γ expression in PBMCs. CONCLUSION: The results suggest a different regulatory action on immune and non-immune tolerogenic mechanisms elicited by protein fraction from different hypoallergenic formulas

Therapeutic Effects of Butyrate on Pediatric Obesity: A Randomized Clinical Trial

Importance: The pediatric obesity disease burden imposes the necessity of new effective strategies. Objective: To determine whether oral butyrate supplementation as an adjunct to standard care is effective in the treatment of pediatric obesity. Design, setting, and participants: A randomized, quadruple-blind, placebo-controlled trial was performed from November 1, 2020, to December 31, 2021, at the Tertiary Center for Pediatric Nutrition, Department of Translational Medical Science, University of Naples Federico II, Naples, Italy. Participants included children aged 5 to 17 years with body mass index (BMI) greater than the 95th percentile. Interventions: Standard care for pediatric obesity supplemented with oral sodium butyrate, 20 mg/kg body weight per day, or placebo for 6 months was administered. Main outcomes and measures: The main outcome was the decrease of at least 0.25 BMI SD scores at 6 months. The secondary outcomes were changes in waist circumference; fasting glucose, insulin, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglyceride, ghrelin, microRNA-221, and interleukin-6 levels; homeostatic model assessment of insulin resistance (HOMA-IR); dietary and lifestyle habits; and gut microbiome structure. Intention-to-treat analysis was conducted. Results: Fifty-four children with obesity (31 girls [57%], mean [SD] age, 11 [2.91] years) were randomized into the butyrate and placebo groups; 4 were lost to follow-up after receiving the intervention in the butyrate group and 2 in the placebo group. At intention-to-treat analysis (n = 54), children treated with butyrate had a higher rate of BMI decrease greater than or equal to 0.25 SD scores at 6 months (96% vs 56%, absolute benefit increase, 40%; 95% CI, 21% to 61%; P < .01). At per-protocol analysis (n = 48), the butyrate group showed the following changes as compared with the placebo group: waist circumference, -5.07 cm (95% CI, -7.68 to -2.46 cm; P < .001); insulin level, -5.41 μU/mL (95% CI, -10.49 to -0.34 μU/mL; P = .03); HOMA-IR, -1.14 (95% CI, -2.13 to -0.15; P = .02); ghrelin level, -47.89 μg/mL (95% CI, -91.80 to -3.98 μg/mL; P < .001); microRNA221 relative expression, -2.17 (95% CI, -3.35 to -0.99; P < .001); and IL-6 level, -4.81 pg/mL (95% CI, -7.74 to -1.88 pg/mL; P < .001). Similar patterns of adherence to standard care were observed in the 2 groups. Baseline gut microbiome signatures predictable of the therapeutic response were identified. Adverse effects included transient mild nausea and headache reported by 2 patients during the first month of butyrate intervention. Conclusions and relevance: Oral butyrate supplementation may be effective in the treatment of pediatric obesity. Trial registration: ClinicalTrials.gov Identifier: NCT04620057

Three-armed RGD-decorated starPLA-PEG nanoshuttle for docetaxel delivery

peer reviewe

The potential role of advanced glycation end products in food allergy pathogenesis

prevalence has dramatically increased in the last two decades. Among dietary factors, it has been hypothesized that advanced glycation endproducts(AGEs), present at high level in junk food, could be involved in FA pathogenesis. AGEs are a heterogeneous group of compounds deriving from sugars(sweets and beverages), autoclaved/processed foods, microwaved foods, more roasted/barbecued meat. To evaluate the AGEs levels in FA children compared with healthy controls and subjects with respiratory allergy. Methods: We evaluated paediatric patients with challenge-proven FA, children with respiratory allergy(RA) and age and sex-matched healthy controls. Subcutaneous AGEs levels were evaluated through the AGE reader. Food-frequency questionnaires were evaluated in all study subjects. In vitro studies were performed on human enterocytes(Caco-2 cells) stimulated with 200 mg/ml of BSA-AGE for 24and48 hours to evaluate effects on gut barrier function: mucin2(mucus production), transpithelial electrical resistance(TEER), ZO-1, occludin expression(intestinal permeability). The direct effects elicited on peripheral blood mononuclear cells (PBMCs) after the treatment with 200 mg/ml of BSA-AGE for 48hours, 4and 7days of treatment were also evaluated. RESULTS: 115 subjects were evaluated and subdivided into 3 groups: group 1 patients with FA (n=31); group 2 patients with RA (n=18), group 3 healthy controls (n=66). The consumption of food containing AGEs was higher in subjects with FA compared to RA children and healthy controls (p<0.05). FA and RA children presented significant higher subcutaneous AGEs levels compared to healthy controls (p<0.05). Linear regression analysis confirmed a significant positive correlation between subcutaneous levels of AGEs and consumption of food containing AGEs. Human enterocytes exposed to BSA-AGE treatment showed a reduction of TEER, of Muc2 and tight junction proteins (Occludin and ZO-1). Moreover, the treatment with BSA-AGE on human PBMCs stimulates pro-inflammatory cytokines TNF-α and Th2 cytokines(IL-5 and IL-13)production , but it was unable to modulate IL-10 production. Finally, after7days of treatment with BSAAGE, we found a low percentage of proliferating CD4+T. CONCLUSIONS: Current hypotheses and models of FA do not adequately explain the dramatic increase observed in the last years

Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.</p

Impact of trans-stent gradient on outcome after PCI: results from a HAWKEYE substudy

To test whether quantitative flow ratio (QFR)-based trans-stent gradient (TSG) is associated with adverse clinical events at follow-up. A post-hoc analysis of the multi-center HAWKEYE study was performed. Vessels post-PCI were divided into four groups (G) as follows: G1: QFR &gt;= 0.90 TSG = 0 (n = 412, 54.8%); G2: QFR &gt;= 0.90, TSG &gt; 0 (n = 216, 28.7%); G3: QFR &lt; 0.90, TSG = 0 (n = 37, 4.9%); G4: QFR &lt; 0.90, TSG &gt; 0 (n = 86, 11.4%). Cox proportional hazards regression model was used to analyze the effect of baseline and prognostic variables. The final reduced model was obtained by backward stepwise variable selection. Receiver operating characteristic (ROC) was plotted and area under the curve (AUC) was calculated and reported. Overall, 449 (59.8%) vessels had a TSG = 0 whereas (40.2%) had TSG &gt; 0. Ten (2.2%) vessel-oriented composite endpoint (VOCE) occurred in vessels with TSG = 0, compared with 43 (14%) in vessels with TSG &gt; 0 (p &lt; 0.01). ROC analysis showed an AUC of 0.74 (95% CI: 0.67 to 0.80; p &lt; 0.001). TSG &gt; 0 was an independent predictor of the VOCE (HR 2.95 [95% CI 1.77-4.91]). The combination of higher TSG and lower final QFR (G4) showed the worst long-term outcome while low TSG and high QFR showed the best outcome (G1) while either high TSG or low QFR (G2, G3) showed intermediate and comparable outcomes. Higher trans-stent gradient was an independent predictor of adverse events and identified a subgroup of patients at higher risk for poor outcomes even when vessel QFR was optimal (&gt; 0.90)