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BackgroundVitamin D is a vital lipophilic vitamin that plays a pivotal role in calcium regulation, bone metabolism, and overall health. It is of the utmost importance to maintain appropriate serum levels of vitamin D from the moment of birth. The recommended daily intake for infants under the age of 12 months is 400 IU. In Europe, vitamin D is available in two forms: as a medicinal product and as a food supplement. The food supplement market is experiencing rapid growth, yet it is characterised by a lack of harmonised regulations, which may give rise to potential risks associated with their widespread use. While food supplements are typically regarded as safe, there is a potential for adverse effects, particularly when dosages are not properly managed.Case report and managementThis report presents the case of a 22-month-old girl who developed nephrocalcinosis as a result of an overdose of vitamin D from a dietary supplement purchased online. The initial presentation was characterised by symptoms such as polydipsia, polyuria and decreased growth. It was subsequently revealed that the child had been receiving an excessively high dose of vitamin D, amounting to 25 times the recommended amount, over a period of seven months. Despite normal calcium levels and renal function at the time of presentation, ultrasound imaging revealed the presence of early-stage nephrocalcinosis. The treatment plan involved hospital admission, intravenous hydration, a thiazide diuretic, potassium citrate, and a low-calcium diet. The vitamin D supplement was ceased. Over the course of a year, the patient demonstrated recovery in growth and normalization of vitamin D levels, although nephrocalcinosis remained stable.ConclusionThis case study highlights the potential dangers of unsupervised vitamin D supplementation, emphasising the importance of healthcare professionals exercising vigilance in prescribing and advising on vitamin D use, particularly in children. Furthermore, it underscores the necessity of establishing a database to track long-term outcomes in paediatric vitamin D intoxication cases, given the rarity of such incidents. This would facilitate the development of appropriate treatment protocols and provide valuable information to parents.

Background: Immune checkpoint inhibitors (ICIs), either alone or in combination with platinum-based chemotherapy, are effective in the first-line treatment of metastatic, non-oncogene-addicted, non-small cell lung cancer (NSCLC). However, when NSCLC patients progress, the efficacy of available treatment options is limited. Methods: We undertook a meta-analysis that compared combination regimens with the current standard of care. Only randomized controlled trials (RCTs) were included (endpoint, overall survival [OS]). Our analysis used an artificial intelligence software program that reconstructs individual patient data from Kaplan–Meier curves. Hazard ratio (HR) with 95% confidence interval (CI) was the main parameter. Heterogeneity was based on Wald’s test and likelihood ratio test. Results: Five RCTs were included, whose experimental arms included five different combinations. In our analysis, these combination regimes showed no OS benefit compared to chemotherapy (HR = 1.066, 95%CI, 0.9311 to 1.221; p = 0.35). Among the five control arms, cross-trial heterogeneity was remarkably low (likelihood ratio test = 3.76 on 4 df, p = 0.40; Wald test = 3.83 on 4 df, p = 0.40. Discussion: In conclusion, five new second-line combination treatments for patients with NSCLC were not found to determine any benefit in terms of OS in comparison with the current standard of care.

Abstract Background In patients with advanced melanoma, immune‐checkpoint inhibitors (ICIs) represent the mainstay for first line treatment. Recently, relatlimab+nivolumab was proposed as a new combination therapy. This review was aimed at summarizing the current data of effectiveness for ICIs. Progression‐free survival (PFS) was the endpoint of our analysis. Methods After a standard literature search, Phase II/III studies comparing different ICI regimens in previously untreated advanced melanoma patients were analyzed. Patient‐level data were reconstructed from Kaplan–Meier curves by application of the IPDfromKM method. These reconstructed datasets were used to perform indirect comparisons between treatments. Standard statistical testing was used, including hazard ratio and medians. A secondary analysis employed the restricted mean survival time. Results Six trials were included in our analysis. Information on PFS from these trials was pooled according to the following treatments: nivolumab or pembrolizumab as monotherapy, or in combination with ipilimumab, and relatlimab + nivolumab. Pembrolizumab+ipilimumab showed significantly better PFS compared with the other treatments; nivolumab+ipilimumab ranked second; the other treatments showed a similar survival pattern. Conclusions The picture of comparative effectiveness resulting from our analysis is complex. The IPDfromKM method is advantageous because it accounts for the length of follow‐up but loses the balance between treatment group and controls determined by randomization. Based on indirect comparisons, the combination of pembrolizumab+ipilimumab showed a particularly high efficacy, and so deserves further investigation. While the effect of between‐trial differences in inclusion criteria plays an important role, our results do not support the proposal of relatlimab+nivolumab as a new standard of care.

Antitumoral drugs (ADs) can induce drug hypersensitivity reactions (DHRs). Rapid drug desensitization (RDD) protocols represent an important option to mitigate recurrent DHRs thus allowing the safe administration of ADs at therapeutic doses. The aim of this retrospective study was to assess the effectiveness of the RDD protocols performed at our institution. The "DESARCh" study was a retrospective, observational study that included consecutive patients who underwent RDD protocols from January 2011 to December 2022 at IRCCS Ospedale Sacro Cuore Don Calabria in Negrar di Valpolicella, Verona, Italy. The RDD protocol consisted of a 5-step protocol with 5 different concentrations of the drugs at 1:1, 1:10, 1:100, 1:1,000 and 1:10,000 dilution given intravenously over a 1-hour infusion each, with concentrations increasing from the most diluted to the most concentrated form, preceded by a 30-min premedication regimen. A total of 66 RDD protocols were administered to 25 female patients with ovarian (64%; n = 16/25), breast (12%; n = 3/25), endometrium (8%; n = 2/25), cervix (8%; n = 2/25), uterine (4%; n = 1/25) and fallopian tubes (4%; n = 1/25) cancers. A known history of atopy/allergy was reported by 36% (n = 9/25) of patients. Patients received RDD protocols because of DHRs to carboplatin (n = 23/66, 34.85%), paclitaxel (n = 18/66, 27.27%), pegylated liposomal doxorubicin (n = 3/66, 4.55%), and trastuzumab (n = 22/66, 33.33%). DHRs were mild-moderate, severe and life-threatening in 60.72%, 28.57% and 10.71% of cases, respectively. The success rate of RDD protocols, defined as the rate of complete administration of full target dose with no breakthrough reactions, was 81.82% (n = 54/66). Success rate was lower for carboplatin compared to other drugs (65.22% vs 90.7%; P =.017678). The RDD protocol used in our institution was found to be safe, with a meaningful success rate. However, further research is needed to better understand the underlying mechanisms of DHRs and to enhance effectiveness, particularly for patients experiencing DHRs to platinum compounds. This study was approved by the ethics committee of Verona and Rovigo (Italy) with approval number 15476 on 10/03/2023 and it was registered with the Register of Observational Studies of the Italian Medicines Agency (AIFA) (available since 31 January 2023), with ID n. 109, on 28/02/2023 (https://www.aifa.gov.it/en/registro-studi-osservazionali). [ABSTRACT FROM AUTHOR]

Background: Immune checkpoint inhibitors (ICIs), either alone or in combination with platinum-based chemotherapy, are effective in the first-line treatment of metastatic, non-oncogene-addicted, non-small cell lung cancer (NSCLC). However, when NSCLC patients progress, the efficacy of available treatment options is limited. Methods: We undertook a meta-analysis that compared combination regimens with the current standard of care. Only randomized controlled trials (RCTs) were included (endpoint, overall survival [OS]). Our analysis used an artificial intelligence software program that reconstructs individual patient data from Kaplan–Meier curves. Hazard ratio (HR) with 95% confidence interval (CI) was the main parameter. Heterogeneity was based on Wald's test and likelihood ratio test. Results: Five RCTs were included, whose experimental arms included five different combinations. In our analysis, these combination regimes showed no OS benefit compared to chemotherapy (HR = 1.066, 95%CI, 0.9311 to 1.221; p = 0.35). Among the five control arms, cross-trial heterogeneity was remarkably low (likelihood ratio test = 3.76 on 4 df, p = 0.40; Wald test = 3.83 on 4 df, p = 0.40. Discussion: In conclusion, five new second-line combination treatments for patients with NSCLC were not found to determine any benefit in terms of OS in comparison with the current standard of care. [ABSTRACT FROM AUTHOR]

AbstractImmune checkpoint inhibitors (ICIs) have revolutionised the treatment of metastatic NSCLC and have become standard first-line therapy both as monotherapy, for patients with PD-L1 expression ≥50%, and in combination with chemotherapy (CT), regardless of PD-L1 expression. This study used an artificial intelligence technique, the IPDfromKM method, to reconstruct individual patient data from Kaplan-Meier curves of phase III randomised clinical trial results to provide a comparative overview of different first-line chemo-immunotherapy options. Overall survival (OS) was estimated using hazard ratios and restricted mean survival time (RMST). Ten clinical trials were included in the analysis. In the squamous population, combinations of cemiplimab + CT (HR = 0.56), pembrolizumab + CT (HR = 0.67), and nivolumab + ipilimumab + CT (HR = 0.71) significantly improved OS compared with CT alone, with no difference between treatments. At longer follow-up, nivolumab + ipilimumab + CT showed longer RMST compared to pembrolizumab + CT in the PD-L1 < 1% subgroup (24.9 months vs. 22.8 months). In non-squamous NSCLC, the survival benefit of ICIs + CT was much more homogeneous, with similar results across the different options. Overall, pembrolizumab + CT showed the best results both in terms of HR (0.68, 95%CI 0.60-0.77) and RMST at long follow-up (30.4 months in the PDL-1 ≥ 1% subgroup and 24 months in the PDL-1 < 1% population). In conclusion, there are some differences between frontline options for treating metastatic NSCLC based on tumour histology and PD-L1 expression. However, further head-to-head trials and longer follow-up are needed to clarify the clinical impact of these differences. [ABSTRACT FROM AUTHOR]

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

Simple Summary: Recently, numerous treatments have been approved for patients with previously untreated locally advanced or metastatic urothelial cancer. These combinations prolong survival compared to chemotherapy, which is generally considered the standard of care. Since direct head-to-head comparisons between these innovative treatments are not available, our analysis was aimed at performing indirect comparisons among these agents, including chemotherapy as a common comparator. In handling these indirect comparisons, our study used an innovative evidence-based method (the IPDfromKM technique) that reconstructs individual patient data from published clinical trials. Overall survival was the endpoint of our analysis. Our results showed that enfortumab vedotin plus pembrolizumab was the most effective treatment at levels of statistical significance. The combinations of a PD(L)-1 inhibitor plus chemotherapy ranked second, while monotherapies with a PD(L)-1 inhibitor ranked third. One strength of this analysis is represented by the large number of different treatments that were evaluated and compared to each other. Background: Patients with locally advanced/metastatic urothelial cancer have been conventionally treated with platinum-based chemotherapy. Recently, numerous new treatments have been proposed to improve overall survival (OS) and reduce adverse effects, but no direct head-to-head comparisons among these agents are available. Methods: The treatments evaluated in our analyses included (a) monotherapy with immune checkpoint inhibitors (ICI); (b) combinations of an ICI with chemotherapy; and (c) combinations of an ICI with other drugs. Using OS as the endpoint, a series of indirect comparisons were performed to rank the most effective regimens against both chemotherapy and each other. Our analysis was based on the application of an artificial intelligence software program (IPDfromKM method) that reconstructs individual patient data from the information reported in the graphs of Kaplan–Meier curves. Results: A total of five studies published in six articles were included. In our main analysis, nivolumab plus chemotherapy showed better OS compared to chemotherapy (HR = 0.70, 95% CI: 0.59–0.82), while durvalumab plus tremelimumab showed no OS benefit (HR = 0.95, 95% CI 0.82–1.11). More interestingly, enfortumab vedotin plus pembrolizumab significantly prolonged OS compared to both chemotherapy alone (HR = 0.53, 95% CI 0.45–0.63) and nivolumab plus chemotherapy (HR = 0.76, 95% CI 0.60–0.97). Discussion and conclusion: Among new treatments for locally advanced and metastatic urothelial cancer, enfortumab vedotin plus pembrolizumab showed the best efficacy in terms of OS. Our results support the use of this combination as a first-line treatment in this setting. [ABSTRACT FROM AUTHOR]

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.

We present a simple, ultra-rapid and robust method to create sharp nanostructures-nanopetals-in a shape memory polymer substrate demonstrating unprecedented enhancements for surface enhanced sensing over large surface areas. These bimetallic nanostructures demonstrate extremely strong surface plasmon resonance effects due to the high density multifaceted petal structures that increase the probability of forming nanogaps. We demonstrate that our nanopetals exhibit extremely strong surface plasmons, confining the emission and enhancing the fluorescence intensity of the nearby high-quantum yield fluorescein by >4000×. The enhancements are confined to the extremely small volumes at the nanopetal borders. This enables us to achieve single molecule detection at relatively high and physiological concentrations. © 2010 American Institute of Physics.

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.

Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease. We describe the use of the recently developed number and brightness method (N&B) that uses confocal images to monitor aggregation of Huntingtin exon 1 protein (Httex1p) directly in living cells. N&B measures the molecular brightness of protein aggregates in the entire cell noninvasively based on intensity fluctuations at each pixel in an image. N&B applied to mutant Httex1p in living cells showed a two-step pathway leading to inclusion formation that is polyQ length dependent and involves four phases. An initial phase of monomer accumulation is followed by formation of small oligomers (5-15 proteins); as protein concentration increases, an inclusion is seeded and forms in the cytoplasm; the growing inclusion recruits most of the Httex1p and depletes the cell leaving only a low concentration of monomers. The behavior of Httex1p in COS-7 and ST14A cells is compared.

JWH-018 and AKB48 are two synthetic cannabinoids (SCBs) belonging to different structural classes and illegally marketed as incense, herbal preparations, or chemical supply for theirs psychoactive cannabis-like effects. Clinical reports from emergency room reported psychomotor agitation as one of the most frequent effects in people assuming SCBs. This study aimed to investigate the psychostimulant properties of JWH-018 and AKB48 in male CD-1 mice and to compare their behavioral and biochemical effects with those caused by cocaine and amphetamine. In vivo studies showed that JWH-018 and AKB48, as cocaine and amphetamine, facilitated spontaneous locomotion in mice. These effects were prevented by CB1 receptor blockade and dopamine (DA) D1/5 and D2/3 receptors inhibition. SPECT-CT studies on dopamine transporter (DAT) revealed that, as cocaine and amphetamine, JWH-018 and AKB48 decreased the [123I]-FP-CIT binding in the mouse striatum. Conversely, in vitro competition binding studies revealed that, unlike cocaine and amphetamine, JWH-018 and AKB48 did not bind to mouse or human DAT. Moreover, microdialysis studies showed that the systemic administration of JWH-018, AKB48, cocaine, and amphetamine stimulated DA release in the nucleus accumbens (NAc) shell of freely moving mice. Finally, unlike amphetamine and cocaine, JWH-018 and AKB48 did not induce any changes on spontaneous [3H]-DA efflux from murine striatal synaptosomes. The present results suggest that SCBs facilitate striatal DA release possibly with different mechanisms than cocaine and amphetamine. Furthermore, they demonstrate, for the first time, that JWH-018 and AKB48 induce a psychostimulant effect in mice possibly by increasing NAc DA release. These data, according to clinical reports, outline the potential psychostimulant action of SCBs highlighting their possible danger to human health.