Whole Exome Sequencing Identifies Epithelial and Immune Dysfunction-Related Biomarkers in Food Protein-Induced Enterocolitis Syndrome.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a food allergy primarily affecting infants, often leading to vomiting and shock. Due to its poorly understood pathophysiology and lack of specific biomarkers, diagnosis is frequently delayed. Understanding FPIES genetics can shed light on disease susceptibility and pathophysiology-key to developing diagnostic, prognostic, preventive and therapeutic strategies. Using a well-characterised cohort of patients we explored the potential genome-wide susceptibility factors underlying FPIES. METHODS: Blood samples from 41 patients with oral food challenge-proven FPIES were collected for a comprehensive whole exome sequencing association study. RESULTS: Notable genetic variants, including rs872786 (RBM8A), rs2241880 (ATG16L1) and rs2289477 (ATG16L1), were identified as significant findings in FPIES. A weighted SKAT model identified six other associated genes including DGKZ and SIRPA. DGKZ induces TGF-ß signalling, crucial for epithelial barrier integrity and IgA production; RBM8A is associated with thrombocytopenia absent radius syndrome, frequently associated with cow's milk allergy; SIRPA is associated with increased neutrophils/monocytes in inflamed tissues as often observed in FPIES; ATG16L1 is associated with inflammatory bowel disease. Coexpression correlation analysis revealed a functional correlation between RBM8A and filaggrin gene (FLG) in stomach and intestine tissue, with filaggrin being a known key pathogenic and risk factor for IgE-mediated food allergy. A transcriptome-wide association study suggested genetic variability in patients impacted gene expression of RBM8A (stomach and pancreas) and ATG16L1 (transverse colon). CONCLUSIONS: This study represents the first case-control exome association study of FPIES patients and marks a crucial step towards unravelling genetic susceptibility factors underpinning the syndrome. Our findings highlight potential factors and pathways contributing to FPIES, including epithelial barrier dysfunction and immune dysregulation. While these results are novel, they are preliminary and need further validation in a second cohort of patients.

Adverse effects of therapeutic phlebotomies: Prospective study of 587 procedures.

BACKGROUND: Therapeutic phlebotomy (TP), a widely used medical procedure, can be performed on diverse patients with iron overload or polyglobulia. However, its adverse events are not well known as most of the information on phlebotomy is derived from healthy blood donors (0.1%-5.3%). In contrast, TP is applicable to a broader, more complex population with comorbidities and old age. To ascertain the incidence of adverse events in phlebotomies, we conducted a prospective study on patients who attended our Unit. STUDY DESIGN AND METHODS: We prospectively gathered data from patients referred to our Unit for TP. Data regarding demographics, health status, and adverse events within at least 24 h of phlebotomy were gathered via a structured questionnaire during each visit. RESULTS: Between August 2021 and September 2022, 189 patients underwent 587 procedures. Most patients were men, over 60 (57.3%) had comorbidities, and 93% underwent at least two procedures during the study period. Twenty patients (10.8%) presented 25 adverse events (4.3% of phlebotomies), usually vasovagal reactions, none of which were clinically relevant, and all were managed by nursing staff on site, with full patient recovery. DISCUSSION: The rate of adverse events (<5%) in patients undergoing TP was low and comparable to that seen in healthy blood donors. Consequently, even old patients and those with some comorbidities can safely undergo TP when the process is carefully managed.

Different prognostic significance of coronary artery and aortic valve calcium in patients with chest pain.

OBJECTIVES: Coronary artery calcification (CorCa) identifies high cardiovascular risk in the general population. In this setting, aortic valve calcification (AoCa) showed contradictory results. Our goal has been to assess the prognostic power of CorCa and AoCa in patients with chest pain who underwent an ECG-gated cardiac multidetector CT (cardiac-MDCT). METHODS: A total of 528 patients without previous known coronary artery disease, with chest pain who underwent a cardiac-MDCT multidetector, were retrospectively recruited. The primary endpoint included death, acute coronary syndrome, stroke, and heart failure. RESULTS: A total of 61 patients (11.6%) had an event during a mean follow-up of almost 6 years (5.95 ± 2.98). The most frequent event was acute coronary syndrome (6.4%). Total mortality was 4.5%. Patients with CorCa > 0 had more events than those without CorCa (17.3% versus 4.3%; p < 0.001). Likewise, when only patients without AoCa were considered (n = 118), clinical events were more frequent in those with CorCa (12.7% versus 3.6%; p = 0.004). After excluding patients with coronary artery disease, events were more frequent in those with CorCa (12.6% versus 4.3%; p = 0.004). The higher the Agatston score, the more frequent the events. Patients with AoCa > 0 had more events than those without (16.5% versus 7.3%; p < 0.001), but in patients without CorCa, no difference in events was seen (6.2% versus 3.6%; p = 0.471). A Cox regression analysis showed age, smoking, prior stroke, and CorCa but not AoCa to be independently related to events. CONCLUSIONS: In summary, CorCa, but not AoCa, is related to cardiovascular events in patients with chest pain who undergo a cardiac-MDCT. CLINICAL RELEVANCE STATEMENT: We show that coronary artery calcification, but not aortic valve calcification, detected in a coronary CT scan is tightly related to cardiovascular events. Although this is a message already shown by other groups in the general population, we do believe that this work is unique because it is restricted to patients with chest pain sent to coronary CT. In other words, our work deals with what we face in our routine everyday practice. KEY POINTS: • The presence and the amount of coronary artery calcification are associated with cardiovascular events in patients with chest pain. • Aortic valve calcification is not associated with cardiovascular events in patients with chest pain.

Human monkeypox virus in a tertiary hospital in Madrid, Spain: An observational study of the clinical and epidemiological characteristics of 53 cases.

A new outbreak of monkeypox virus (MPXV) infection, a zoonotic infection endemic in Central and West Africa, is spreading throughout the world with new epidemiology and clinical features. Our aim was to characterize patients presenting to Dermatology emergency room with a MPXV infection between 15 May and 30 June 2022 in a tertiary hospital in Madrid, Spain. We collected 53 patients and describe their clinical, demographic and epidemiological characteristics and followed their evolution. Most of the patients were men who had sex with men with high-risk sexual practices and no recent travels abroad. Most of them (91%) had had a sexually transmitted infection before. All patients had typical skin lesions consisting of vesicular-pustular rash with central umbilication which was localized or disseminated. The most frequent extracutaneous symptoms were fever, painful regional lymphadenopathy and asthenia. Proctitis was present in more than one third of patients. All patients were diagnosed by real time polymerase chain reaction of samples obtained from skin lesions. Pharyngeal and/or rectal exudates demonstrated MPXV in 74% of patients. The current worldwide outbreak of MPXV infections shows epidemiological and clinical differences from previous ones. Clinicians should be aware of these characteristics to correctly diagnose this emerging disease.

Clinical, biochemical and genetic characteristics of MOGS-CDG: a rare congenital disorder of glycosylation.

PURPOSE: To summarise the clinical, molecular and biochemical phenotype of mannosyl-oligosaccharide glucosidase-related congenital disorders of glycosylation (MOGS-CDG), which presents with variable clinical manifestations, and to analyse which clinical biochemical assay consistently supports diagnosis in individuals with bi-allelic variants in MOGS. METHODS: Phenotypic characterisation was performed through an international and multicentre collaboration. Genetic testing was done by exome sequencing and targeted arrays. Biochemical assays on serum and urine were performed to delineate the biochemical signature of MOGS-CDG. RESULTS: Clinical phenotyping revealed heterogeneity in MOGS-CDG, including neurological, immunological and skeletal phenotypes. Bi-allelic variants in MOGS were identified in 12 individuals from 11 families. The severity in each organ system was variable, without definite genotype correlation. Urine oligosaccharide analysis was consistently abnormal for all affected probands, whereas other biochemical analyses such as serum transferrin analysis was not consistently abnormal. CONCLUSION: The clinical phenotype of MOGS-CDG includes multisystemic involvement with variable severity. Molecular analysis, combined with biochemical testing, is important for diagnosis. In MOGS-CDG, urine oligosaccharide analysis via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry can be used as a reliable biochemical test for screening and confirmation of disease.

Chemotherapy de-escalation using an 18F-FDG-PET-based pathological response-adapted strategy in patients with HER2-positive early breast cancer (PHERGain): a multicentre, randomised, open-label, non-comparative, phase 2 trial.

BACKGROUND: Several de-escalation approaches are under investigation in patients with HER2-positive, early-stage breast cancer. We assessed early metabolic responses to neoadjuvant trastuzumab and pertuzumab using 18F-fluorodeoxyglucose (18F-FDG)-PET (18F-FDG-PET) and the possibility of chemotherapy de-escalation using a pathological response-adapted strategy. METHODS: We did a multicentre, randomised, open-label, non-comparative, phase 2 trial in 45 hospitals in Spain, France, Belgium, Germany, the UK, Italy, and Portugal. Eligible participants were women aged 18 years or older with centrally confirmed, HER2-positive, stage I-IIIA, invasive, operable breast cancer (≥1·5 cm tumour size) with at least one breast lesion evaluable by 18F-FDG-PET, an Eastern Cooperative Oncology Group performance status of 0 or 1, and a baseline left ventricular ejection fraction of at least 55%. We randomly assigned participants (1:4), via an interactive response system using central block randomisation with block sizes of five, stratified by hormone receptor status, to either docetaxel (75 mg/m2 intravenous), carboplatin (area under the concentration-time curve 6 mg/mL per min intravenous), trastuzumab (subcutaneous 600 mg fixed dose), and pertuzumab (intravenous 840 mg loading dose, 420 mg maintenance doses; group A); or trastuzumab and pertuzumab (group B). Hormone receptor-positive patients allocated to group B were additionally given letrozole if postmenopausal (2·5 mg/day orally) or tamoxifen if premenopausal (20 mg/day orally). Centrally reviewed 18F-FDG-PET scans were done before randomisation and after two treatment cycles. Patients assigned to group A completed six cycles of treatment (every 3 weeks) regardless of 18F-FDG-PET results. All patients assigned to group B initially received two cycles of trastuzumab and pertuzumab. 18F-FDG-PET responders in group B continued this treatment for six further cycles; 18F-FDG-PET non-responders in this group were switched to six cycles of docetaxel, carboplatin, trastuzumab, and pertuzumab. Surgery was done 2-6 weeks after the last dose of study treatment. Adjuvant treatment was selected according to the neoadjuvant treatment administered, pathological response, hormone receptor status, and clinical stage at diagnosis. The coprimary endpoints were the proportion of 18F-FDG-PET responders in group B with a pathological complete response in the breast and axilla (ypT0/is ypN0) as determined by a local pathologist after surgery after eight cycles of treatment, and 3-year invasive disease-free survival of patients in group B, both assessed by intention to treat. The definitive assessment of pathological complete response was done at this primary analysis; follow-up to assess invasive disease-free survival is continuing, hence these data are not included in this Article. Safety was assessed in all participants who received at least one dose of study drug. Health-related quality-of-life was assessed with EORTC QLQ-C30 and QLQ-BR23 questionnaires at baseline, after two cycles of treatment, and before surgery. This trial is registered with EudraCT (2016-002676-27) and ClinicalTrials.gov (NCT03161353), and is ongoing. FINDINGS: Between June 26, 2017, and April 24, 2019, we randomly assigned 71 patients to group A and 285 to group B. Median follow-up was 5·7 months (IQR 5·3-6·0). 227 (80%) of 285 patients in group B were 18F-FDG-PET responders, of whom 86 (37·9%, 95% CI 31·6-44·5; p<0·0001 compared with the historical rate) of 227 had a pathological complete response. The most common haematological grade 3-4 adverse events were anaemia (six [9%] of 68 patients in group A vs four [1%] of 283 patients in group B), neutropenia (16 [24%] vs ten [4%]), and febrile neutropenia (14 [21%] vs 11 [4%]). Serious adverse events occurred in 20 (29%) of 68 patients in group A versus 13 (5%) of 283 patients in group B. No deaths were reported during neoadjuvant treatment. Global health status declined by at least 10% in 65·0% (95% CI 46·5-72·4) and 35·5% (29·7-41·7) of patients in groups A and B, respectively INTERPRETATION: 18F-FDG-PET identified patients with HER2-positive, early-stage breast cancer who were likely to benefit from chemotherapy-free dual HER2 blockade with trastuzumab and pertuzumab, and a reduced impact on global health status. Depending on the forthcoming results for the 3-year invasive disease-free survival endpoint, this strategy might be a valid approach to select patients not requiring chemotherapy. FUNDING: F Hoffmann-La Roche.

A phase Ib/II study of xentuzumab, an IGF-neutralising antibody, combined with exemestane and everolimus in hormone receptor-positive, HER2-negative locally advanced/metastatic breast cancer.

BACKGROUND: Xentuzumab-a humanised IgG1 monoclonal antibody-binds IGF-1 and IGF-2, inhibiting their growth-promoting signalling and suppressing AKT activation by everolimus. This phase Ib/II exploratory trial evaluated xentuzumab plus everolimus and exemestane in hormone receptor-positive, locally advanced and/or metastatic breast cancer (LA/MBC). METHODS: Patients with hormone receptor-positive/HER2-negative LA/MBC resistant to non-steroidal aromatase inhibitors were enrolled. Maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of xentuzumab/everolimus/exemestane were determined in phase I (single-arm, dose-escalation). In phase II (open-label), patients were randomised 1:1 to the RP2D of xentuzumab/everolimus/exemestane or everolimus/exemestane alone. Randomisation was stratified by the presence of visceral metastases. Primary endpoint was progression-free survival (PFS). RESULTS: MTD was determined as xentuzumab 1000 mg weekly plus everolimus 10 mg/day and exemestane 25 mg/day. A total of 140 patients were enrolled in phase II (70 to each arm). Further recruitment was stopped following an unfavourable benefit-risk assessment by the internal Data Monitoring Committee appointed by the sponsor. Xentuzumab was discontinued; patients could receive everolimus/exemestane if clinically indicated. Median PFS was 7.3 months (95% CI 3.3-not calculable) in the xentuzumab/everolimus/exemestane group and 5.6 months (3.7-9.1) in the everolimus/exemestane group (hazard ratio 0.97, 95% CI 0.57-1.65; P = 0.9057). In a pre-specified subgroup of patients without visceral metastases at screening, xentuzumab/everolimus/exemestane showed evidence of PFS benefit versus everolimus/exemestane (hazard ratio 0.21 [0.05-0.98]; P = 0.0293). Most common any-cause adverse events in phase II were diarrhoea (29 [41.4%] in the xentuzumab/everolimus/exemestane group versus 20 [29.0%] in the everolimus/exemestane group), mucosal inflammation (27 [38.6%] versus 21 [30.4%]), stomatitis (24 [34.3%] versus 24 [34.8%]), and asthenia (21 [30.0%] versus 24 [34.8%]). CONCLUSIONS: Addition of xentuzumab to everolimus/exemestane did not improve PFS in the overall population, leading to early discontinuation of the trial. Evidence of PFS benefit was observed in patients without visceral metastases when treated with xentuzumab/everolimus/exemestane, leading to initiation of the phase II XENERA™-1 trial (NCT03659136). TRIAL REGISTRATION: ClinicalTrials.gov, NCT02123823 . Prospectively registered, 8 March 2013.

Improvement of Phase Change Materials (PCM) Used for Solar Process Heat Applications.

The high intermittency of solar energy is still a challenge yet to be overcome. The use of thermal storage has proven to be a good option, with phase change materials (PCM) as very promising candidates. Nevertheless, PCM compounds have typically poor thermal conductivity, reducing their attractiveness for commercial uses. This paper demonstrates the viability of increasing the PCM effective thermal conductivity to industrial required values (around 4 W/m·K) by using metal wool infiltrated into the resin under vacuum conditions. To achieve this result, the authors used an inert resin, decoupling the specific PCM material selection from the enhancement effect of the metal wools. To ensure proper behavior of the metal wool under standard industrial environments at a broad range of temperatures, a set of analyses were performed at high temperatures and an inert atmosphere, presenting a thorough analysis of the obtained results.

The OLYMPUS Architecture-Oblivious Identity Management for Private User-Friendly Services.

Privacy enhancing technologies (PETs) allow to achieve user's transactions unlinkability across different online Service Providers. However, current PETs fail to guarantee unlinkability against the Identity Provider (IdP), which becomes a single point of failure in terms of privacy and security, and therefore, might impersonate its users. To address this issue, OLYMPUS EU project establishes an interoperable framework of technologies for a distributed privacy-preserving identity management based on cryptographic techniques that can be applied both to online and offline scenarios. Namely, distributed cryptographic techniques based on threshold cryptography are used to split up the role of the Identity Provider (IdP) into several authorities so that a single entity is not able to impersonate or track its users. The architecture leverages PET technologies, such as distributed threshold-based signatures and privacy attribute-based credentials (p-ABC), so that the signed tokens and the ABC credentials are managed in a distributed way by several IdPs. This paper describes the Olympus architecture, including its associated requirements, the main building blocks and processes, as well as the associated use cases. In addition, the paper shows how the Olympus oblivious architecture can be used to achieve privacy-preserving M2M offline transactions between IoT devices.

First steps in the formulation of praziquantel nanosuspensions for pharmaceutical applications.

Praziquantel (PZQ), a broad spectrum anthelmintic drug, cannot be found in acceptable dosage forms for elderly patients, paediatric patients, and for veterinary use. In fact, very little has been done up to now in the formulation of liquid dosage forms, being they always formulated for parenteral administration. To beat this important challenge, it was accomplished a comprehensive analysis of the influence of two elementary physicochemical aspects, i.e. surface thermodynamic and electrokinetic properties, on the colloidal stability of PZQ nanosuspensions. The hydrophobic character of the drug, intensely determining the flocculation curves, was confirmed by the thermodynamic characterization. The electrophoretic characterization, in combination with the sedimentation and relative absorbance versus time curves, highlighted that the electrical double layer thickness and the surface charge can play an essential role in the stability of the pharmaceutical colloid. Finally, it was demonstrated that controlling the pH values and the incorporation of electrolytes can help in formulating PZQ aqueous nanosuspensions with appropriate stability and redispersibility behaviours for pharmaceutical use.

Oral metronomic vinorelbine combined with endocrine therapy in hormone receptor-positive HER2-negative breast cancer: SOLTI-1501 VENTANA window of opportunity trial.

BACKGROUND: The biological effect of oral metronomic vinorelbine (mVNB) alone or in combination with endocrine therapy in patients with hormone receptor-positive (HR+)/HER2-negative breast cancer has been scarcely addressed. METHODS: Postmenopausal women with untreated stage I-III HR+/HER2-negative breast cancer were randomized (1:1:1) to receive 3 weeks of letrozole (LTZ) 2.5 mg/day, oral mVNB 50 mg 3 days/week, or the combination. The primary objective was to evaluate, within PAM50 Luminal A/B disease, if the anti-proliferative effect of LTZ+mVNB was superior to monotherapy. An anti-proliferative effect was defined as the mean relative decrease of the PAM50 11-gene proliferation score in combination arm vs. both monotherapy arms. Secondary objectives included the evaluation of a comprehensive panel of breast cancer-related genes and safety. An unplanned analysis of stromal tumor-infiltrating lymphocytes (sTILs) was also performed. PAM50 analyses were performed using the nCounter®-based Breast Cancer 360™ gene panel, which includes 752 genes and 32 signatures. RESULTS: Sixty-one patients were randomized, and 54 paired samples (89%) were analyzed. The main patient characteristics were mean age of 67, mean tumor size of 1.7 cm, mean Ki67 of 14.3%, stage I (55.7%), and grades 1-2 (90%). Most baseline samples were PAM50 Luminal A (74.1%) or B (22.2%). The anti-proliferative effect of 3 weeks of LTZ+mVNB (- 73.2%) was superior to both monotherapy arms combined (- 49.9%; p = 0.001) and mVNB (- 19.1%; p < 0.001). The anti-proliferative effect of LTZ+mVNB (- 73.2%) was numerically higher compared to LTZ (- 65.7%) but did not reach statistical significance (p = 0.328). LTZ+mVNB induced high expression of immune-related genes and gene signatures, including CD8 T cell signature and PDL1 gene and low expression of ER-regulated genes (e.g., progesterone receptor) and cell cycle-related and DNA repair genes. In tumors with ≤ 10% sTILs at baseline, a statistically significant increase in sTILs was observed following LTZ (paired analysis p = 0.049) and LTZ+mVNB (p = 0.012). Grade 3 adverse events occurred in 3.4% of the cases. CONCLUSIONS: Short-term mVNB is well-tolerated and presents anti-proliferative activity alone and in combination with LTZ. The high expression of immune-related biological processes and sTILs observed with the combination opens the possibility of studying this combination with immunotherapy. Further investigation comparing these biological results with other metronomic schedules or drug combinations is warranted. TRIAL REGISTRATION: NCT02802748 , registered 16 June 2016.

A Gene Homologous to rRNA Methylase Genes Confers Erythromycin and Clindamycin Resistance in Bifidobacterium breve.

Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island.IMPORTANCEBifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains.

Aquaporin 11 is related to cryotolerance and fertilising ability of frozen-thawed bull spermatozoa.

Aquaporins (AQPs) are channel proteins involved in the transport of water and solutes across biological membranes. In the present study we identified and localised aquaporin 11 (AQP11) in bull spermatozoa and investigated the relationship between the relative AQP11 content, sperm cryotolerance and the fertilising ability of frozen-thawed semen. Bull ejaculates were classified into two groups of good and poor freezability and assessed through immunofluorescence and immunoblotting analyses before and after cryopreservation. AQP11 was localised throughout the entire tail and along the sperm head. These findings were confirmed through immunoblotting, which showed a specific band of approximately 50 kDa corresponding to AQP11. The relative amount of AQP11 was significantly (P<0.05) higher in both fresh and frozen-thawed spermatozoa from bull ejaculates with good freezability compared with those with poorer freezability. In addition, in vitro oocyte penetration rates and non-return rates 56 days after AI were correlated with the relative AQP11 content in fresh spermatozoa. In conclusion, AQP11 is present in the head and tail of bull spermatozoa and its relative amount in fresh and frozen-thawed spermatozoa is related to the resilience of the spermatozoa to withstand cryopreservation and the fertilising ability of frozen-thawed spermatozoa. Further research is needed to elucidate the actual role of sperm AQP11 in bovine fertility.

HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer (PAMELA): an open-label, single-group, multicentre, phase 2 trial.

BACKGROUND: HER2-positive breast cancer consists of four intrinsic molecular subtypes-luminal A, luminal B, HER2-enriched, and basal-like-and a normal-like subtype, with the HER2-enriched subtype having the highest activation of the EGFR-HER2 pathway. We aimed to test the hypothesis that patients with the HER2-enriched subtype benefit the most from dual HER2 blockade. METHODS: PAMELA is an open-label, single-group, phase 2 trial done in 19 hospitals in Spain. We recruited female patients aged at least 18 years with previously untreated, centrally confirmed HER2-positive, stage I-IIIA invasive breast cancer regardless of hormone receptor status. Patients were given lapatinib (1000 mg per day orally) and trastuzumab (loading dose of 8 mg/kg, followed by 6 mg/kg every 3 weeks intravenously) for 18 weeks; hormone receptor-positive patients were additionally given letrozole (2·5 mg per day orally; if menopausal) or tamoxifen (20 mg per day orally; if premenopausal). Surgery was done 1-3 weeks after the last dose of study treatment. Intrinsic molecular subtypes of tumour biopsy samples taken at baseline (day 0) and day 14 were determined with the PAM50 predictor. The primary outcome was the ability of the HER2-enriched subtype to predict pathological complete response at the time of surgery. The primary outcome was assessed in the evaluable population (ie, all patients who had initial tumour biopsy samples available and who underwent definitive surgery) and safety was assessed in all patients who received at least one part of study treatment. This study is registered with ClinicalTrials.gov, number NCT01973660, and is completed. FINDINGS: Between Oct 28, 2013, and Nov 26, 2015, we recruited 151 patients, of whom 14 (9%) discontinued treatment and 137 (91%) completed treatment as planned. At baseline, most patients had the HER2-enriched subtype (101 [67%]), followed by luminal A (22 [15%]), luminal B (16 [11%]), basal-like (nine [6%]), and normal-like (three [2%]) subtypes. At the time of surgery, 46 (30%, 95% CI 23-39) of 151 patients had pathological complete response in the breast. 41 (41%, 31-51) of 101 patients with the HER2-enriched subtype and five (10%, 4-23) of 50 patients with non-HER2-enriched subtypes achieved pathological complete response at the time of surgery (odds ratio 6·2, 95% CI 2·3-16·8; p=0·0004). INTERPRETATION: The HER2-enriched subtype can identify patients with HER2-positive breast cancer who are likely to benefit from dual HER2 blockade therapies. FUNDING: GlaxoSmithKline, Susan Komen Foundation, CERCA Programme-Generalitat de Catalunya, Banco Bilbao Vizcaya Argentaria Foundation, Pas a Pas, and the Breast Cancer Research Foundation.

Aquaporins in boar spermatozoa. Part II: detection and localisation of aquaglyceroporin 3.

The proteins belonging to the aquaporin family play a fundamental role in water and solute transport across biological membranes. While the presence of these proteins has been extensively studied in somatic cells, their function in mammalian spermatozoa has been studied less. The present study was designed to identify and localise aquaglyceroporin 3 (AQP3) in boar spermatozoa. With this purpose, 29 fresh ejaculates from post-pubertal Piétrain boars were classified into two groups based upon their sperm quality and subsequently evaluated through western blot and immunofluorescence assessments. Western blotting showed the specific signal band of AQP3 at 25 kDa, whereas immunofluorescence assessments allowed us to identify two different AQP3 localisation patterns: (1) spermatozoa presenting a clear labelling located only in the mid-piece and (2) spermatozoa exhibiting a distribution pattern in the head and along the entire tail. The first staining pattern was predominant in all studied ejaculates. Despite individual differences in AQP3 content and localisation between boar ejaculates, these differences were not correlated with sperm quality. In conclusion, although AQP3 is present in boar spermatozoa in two different localisation patterns, neither the AQP3 content nor its localisation have been found to be associated with conventional sperm parameters.

Aquaglyceroporins 3 and 7 in bull spermatozoa: identification, localisation and their relationship with sperm cryotolerance.

The present study aimed to determine the localisation of aquaglyceroporins 3 (AQP3) and 7 (AQP7) in bull spermatozoa and their relationship with the sperm cell's resilience to withstand cryopreservation (i.e. cryotolerance). A total of 18 bull ejaculates were cryopreserved and their sperm quality analysed before and after freeze-thawing. The presence and localisation of AQP3 and AQP7 was determined through immunoblotting and immunocytochemistry. AQP3 was found in the mid-piece and AQP7 in the mid-piece and post-acrosomal region of bull spermatozoa. Immunoblotting showed specific signal bands at 30 and 60kDa for AQP3 and at 25kDa for AQP7. Neither the relative abundance of AQP3 and AQP7 nor their localisation patterns was altered by cryopreservation but individual differences between bull ejaculates were found in immunoblots. In order to determine whether these individual differences were related to sperm cryotolerance, bull ejaculates were classified as having good (GFE) or poor freezability (PFE) on the basis of their sperm quality after thawing. While the relative abundance of AQP3 before cryopreservation did not differ between ejaculates with GFE and PFE, the abundance of AQP7 was higher in GFE than in PFE ejaculates. This finding was further confirmed through principal component and linear regression analyses. In conclusion, the relative abundance of AQP7 in fresh semen may be used as a marker to predict bull sperm cryotolerance.

Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality.

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

A proteomic approach towards understanding the cross talk between Bacteroides fragilis and Bifidobacterium longum in coculture.

A better understanding of the interactions among intestinal microbes is needed to decipher the complex cross talk that takes place within the human gut. Bacteroides and Bifidobacterium genera are among the most relevant intestinal bacteria, and it has been previously reported that coculturing of these 2 microorganisms affects their survival. Therefore, coculturing of Bifidobacterium longum NB667 and Bacteroides fragilis DSMZ2151 was performed with the aim of unravelling the mechanisms involved in their interaction. To this end, we applied proteomic (2D-DIGE) analyses, and by chromatographic techniques we quantified the bacterial metabolites produced during coincubation. Coculture stimulated the growth of B. longum, retarding that of B. fragilis, with concomitant changes in the production of some proteins and metabolites of both bacteria. The combined culture promoted upregulation of the bifidobacterial pyruvate kinase and downregulation of the Bacteroides phosphoenolpyruvate carboxykinase - 2 enzymes involved in the catabolism of carbohydrates. Moreover, B. fragilis FKBP-type peptidyl-prolyl cis-trans isomerase, a protein with chaperone-like activity, was found to be overproduced in coculture, suggesting the induction of a stress response in this microorganism. This study provides mechanistic data to deepen our understanding of the interaction between Bacteroides and Bifidobacterium intestinal populations.

Standard versus continuous administration of capecitabine in metastatic breast cancer (GEICAM/2009-05): a randomized, noninferiority phase II trial with a pharmacogenetic analysis.

BACKGROUND: The approved capecitabine regimen as monotherapy in metastatic breast cancer (MBC) is 1,250 mg/m(2) twice daily for 2 weeks on and 1 week off (Cint). Dose modifications are often required because of severe hand-foot syndrome (HFS). We tested a continuous regimen with a lower daily dose but a similar cumulative dose in an attempt to reduce the severity of adverse events (AEs) while maintaining efficacy. METHODS: We randomized 195 patients with HER-2/neu-negative MBC to capecitabine 800 mg/m(2) twice daily throughout the 21-day cycle (Ccont) or to Cint to assess noninferiority in the percentage of patients free of progression at 1 year. Secondary endpoints included efficacy and safety. Associations between polymorphisms in capecitabine metabolism-related genes and drug response were assessed. RESULTS: The percentage of patients free of progression at 1 year was 27.3% with Cint versus 25.3% with Ccont (difference of -2.0%; 95% confidence interval: -15.5% to 11.5%, exceeding the 15% deemed noninferior). Differences regarding other efficacy variables were also not found. Grade 3-4 HFS was the most frequent AE (41.1% in Cint vs. 42.3% in Ccont). Grade 3-4 neutropenia, thrombocytopenia, diarrhea, and stomatitis were more frequent with Cint. A 5' untranslated region polymorphism in the carboxylesterase 2 gene was associated with HFS. One polymorphism in cytidine deaminase and two in thymidine phosphorylase were associated with survival. CONCLUSION: Our study was unable to show noninferiority with the continuous capecitabine regimen (Ccont) compared with the approved intermittent regimen (Cint). Further investigation is required to improve HFS. Polymorphisms in several genes might contribute to interindividual differences in response to capecitabine.