Clinical Therapeutic Treatment Research Articles

O-304 Whole-exome sequencing in women with reproductive failure is a potentially useful diagnostic test in clinical practice

Abstract Study question Could whole-exome sequencing (WES) be useful in clinical practice for primary infertile female involving oocyte or embryo defects and unexplained recurrent miscarriage (RM) patients? Summary answer Identifying key genes involved in oocyte and embryo development helps explain approximately 17% cases of reproductive failure. WES results will help optimize clinical therapeutic treatment. What is known already Dozens of genes have been reported to be the genetic causes of human infertility. Variants in these genes have specific effects on certain processes of human early reproduction and result in a spectrum of phenotypes, such as oocyte maturation arrest, fertilization defects, cleavage failure, early embryonic lethality, and recurrent miscarriage. Study design, size, duration This retrospective cohort study was conducted from July 2020 to August 2022. A total of 129 affected females were enrolled. Most of them were primary infertile women who suffered repeated failure of in vitro fertilization (≥2 cycles) due to abnormal development of oocytes or embryos (PI group, n = 125). Our cohort also comprised a small number of unexplained RM subjects without offspring (RM group, n = 4). All patients and their spouses had normal karyotype. Participants/materials, setting, methods The peripheral blood samples from all the participants and all of their available family members were obtained for DNA extraction. Genomic DNA samples from the probands were subjected to WES to identify candidate variants. Subsequently, the variants were validated by Sanger sequencing or qPCR. Familial co-segregation analyses were then carried out within the family members. The relationship between phenotype and genotype by clinical tests and the clinical records of the patients was studied. Main results and the role of chance Firstly, variants that are potentially relevant to the phenotypes of female infertility were identified in 17.05% of cases (n = 22), including TUBB8 in 14 cases, PATL2 in 2 cases, ZP2, ZP3, PANX1, TLE6, NLRP2 and NLRP7 in 1 case for each. A breakdown by phenotype revealed that we identified variants in 16.00% (n = 20) of PI group and 50% (n = 2) of RM group. Secondly, 16 TUBB8 variants were identified in 14 cases, of which 8 were novel, 3 were previously reported, and 5 were novel amino acid change occurring at the same position as another previously reported change. We found 3 TUBB8 variants (p.C211R, p.T232I, p.A342T) in 3 embryonic arrest families and 1 TUBB8 mutation (p.A102V) in a RM family, which were maternally inherited. Thirdly, 75 patients in PI group without phenotypic variant underwent ≥1 cycles of ovulation and/or frozen embryo transfer after WES, and 49.33% had achieved clinical pregnancy (n = 37). Limitations, reasons for caution The genomic DNA of parents were not available in all these 22 families, so we cannot determine whether the variant is de novo or inherited in some cases. Additionally, further functional studies should be performed to prove that these novel mutations affect protein function. Wider implications of the findings The present study expands the kinds of variants and phenotypic spectrum of gene mutations with regard to female reproductive failure. Genetic tests for causative genes involved in oocyte and embryo defects were recommended for primary infertile patients who suffered from these conditions and for unexplained RM patients. Trial registration number not applicable

Mechanisms Underlying Exercise-Induced BDNF Stimulated Memory Improvements in Rodents

Introduction: Increases in Brain-Derived Neurotrophic Factor (BDNF), are known to be associated with improvements in memory and can be increased by exercise. This suggests that these memory improvements could be induced from exercise associated increases in BDNF in key memory related areas of the brain such as the hippocampus. However, the underlying neurobiology outlining how exercise may affect memory remains elusive. Thus, the focus of this review is to understand the relationship between exercise induced increases in BDNF and memory improvements, and to identify potential pathways of activation that may induce these effects. Methods: An in-depth literature review was performed to analyze the mechanisms of memory involving exercise induced increases of BDNF in rodents. Both primary and review articles were used, found through Google Scholar, PubMed, and the George Washington University Gelman Library databases, and were found using search terms that included exercise, memory, rodents, and BDNF. Results: Pathways found to be potentially involved in this mechanism include the TrkB ERK1 pathway, TrkB activation of β-CaMKII, PI3-K and MAPK pathways, and the BDNF/TrkB/SYN pathway, with the SIRT1/PGC1a/FNDC5 pathway being a potential upstream pathway to induce BDNF activity. It was also found that exercise induced BDNF increases can recover memory impairments in different neurodegenerative states, such as in rodent models of Alzheimer’s disease and Down syndrome. Discussion: The collected studies demonstrate that exercise induced increases in BDNF expression are related to improved memory at least partially due to increased BDNF expression in memory associated brain regions, such as the hippocampus. The underlying neurobiology is still unclear, although potential pathways for this mechanism have been identified. Conclusion: These results of this review expose avenues of research to better understand the underlying neurobiology of exercise enhanced BDNF effects on memory improvements. The identified link between exercise and memory improvements via exercise induced BDNF increases can be potentially applied and used for future investigations into clinical therapeutic treatments for improving memory through exercise.

Acinetobacter baumannii: Its Clinical Significance in Human and Veterinary Medicine.

Acinetobacter baumannii is a Gram-negative, opportunistic pathogen, causing severe infections difficult to treat. The A. baumannii infection rate has increased year by year in human medicine and it is also considered as a major cause of nosocomial infections worldwide. This bacterium, also well known for its ability to form biofilms, has a strong environmental adaptability and the characteristics of multi-drug resistance. Indeed, strains showing fully resistant profiles represent a worrisome problem in clinical therapeutic treatment. Furthermore, A. baumannii-associated veterinary nosocomial infections has been reported in recent literature. Particularly, carbapenem-resistant A. baumannii can be considered an emerging opportunistic pathogen in human medicine as well as in veterinary medicine.

Highly Enhanced Antitumor Immunity by a Three-Barreled Strategy of the l-Arginine-Promoted Nanovaccine and Gene-Mediated PD-L1 Blockade.

Weak T cell responses and immune checkpoints within tumors could be two key factors for limiting antitumor efficacy in the field of cancer immunotherapy. Thus, the combined strategy of tumor vaccines and immune checkpoint blockade has been widely studied and expected to boost antitumor immune responses. Herein, we first developed a two-barreled strategy to combine the nanovaccine with a gene-mediated PD-L1 blockade. On the one hand, polyethyleneimine (PEI) worked as a vaccine carrier to codeliver the antigen ovalbumin (OVA) and the adjuvant unmethylated cytosine-phosphate-guanine (CpG) to formulate the PEI/OVA/CpG nanovaccine through electrostatic binding, which realized both dendritic cell activation and antigen cross-presentation enhancement. On the other hand, the PD-L1 silence gene was loaded by PEI to form PEI/pshPD-L1 complexes, which were further in situ shielded by aldehyde-modified polyethylene glycol (OHC-PEG-CHO) via pH-responsive Schiff base bonds. The formed pshPD-L1@NPs could decrease PD-L1 expression on the tumor cells. However, such a combined two-barreled strategy improved feebly for tumor inhibition in comparison with monotherapy, exhibiting the antagonistic effect, which might be due to the limited T cell response enhancement in the tumor microenvironment. To solve this problem, we have further developed a three-barreled strategy to combine oral administration of l-arginine, which worked as an amplifier to induce robust T cell response enhancement, without causing the upregulation of other negative immune regulators. Superior antitumor behavior and tumor rechallenge protection were realized by the three-barreled strategy in B16F10-OVA (B16-OVA)-bearing mice. The unique three-barreled strategy we developed might offer a novel clinical therapeutic treatment.

141 - Increased proliferation/aggressiveness and metastatic potential of therapy targeted surviving breast adenocarcinoma cells as well as bystander non-targeted cancer cells; role of therapy-induced iNOS/NO, and beneficial effects of iNOS inhibitors

We have recently uniquely shown that human breast cancer cells (adenocarcinoma MDA-MB 231), exploit endogenous nitric oxide (NO) to resist elimination by photodynamic therapy (PDT), cisplatin (CDDP) chemotherapy or combination of both. Inducible nitric oxide synthase (iNOS) was rapidly and persistently upregulated after the treatment challenge, and the resulting NO signaled not only for greater resistance, but also greater growth, migration, and invasion aggressiveness of not only cancer cells surviving the challenge, but also bystander cells which escaped initial therapy. Ionizing radiation of specifically targeted cells in a given population is known to elicit pro-death or pro-survival responses in non-targeted bystander cells, but far less is known about such effects in the case of non-ionizing therapies. We will report on our most recent studies testing hypothesis that photodynamically or pharmacologically stressed breast adenocarcinoma cells can elicit NO-mediated pro-growth/migration aggressive responses not only in surviving target cells but also in non-stressed bystander cells. Switching to a more aggressive phenotype during clinical therapeutic treatment (either PDT or chemo/PDT) would be an alarming prospect because it might lead to greater cancer metastatic spread. We will also report on our attempts to overcome/minimize such effects by using classical inhibitors of iNOS activity (s.a. 1400W) and recently introduced bromodomain and extra-terminal domain (BET) transcriptional inhibitors (s.a. JQ1). (Supported by NCN grant 2017/27/B/NZ5/02620).

Prospect of Combination and Sequential Therapies for Postmenopausal Osteoporosis.

Osteoporosis is a systemic bone disease, which is associated with the osteoporotic fracture. Along with prolonged life expectancy, the fracture risk increases dramatically with age. So far, it is usually the first choice to using antiresorptive agents. However, current clinical therapeutic treatments are not enough to inhibit the long-term bone loss and the rising risk of bone fractures. Recently, combination and sequential therapies with anti-osteoporosis drugs have become an inevitable trend. The purpose of this review is to summarize the present knowledge related to the combination and sequential therapies with anti-osteoporosis drugs. All articles published in PubMed until 2016 on the combination and sequential therapies with antiosteoporosis drugs were reviewed and the relevant literature was described in this review. As the first-line therapy for osteoporosis, the long-term safety and efficacy on bisphosphonate, teriparatide, and new anti-osteoporosis drugs are ongoing concerns. Combination or sequential therapies of boneforming agents and anti-resorptive agents, or two kinds of anti-resorptive agents, or strontium ranelate and other anti-osteoporosis agents have the advantages on BMD compared with monotherapy. So far, it is still an important public health strategy to therapy on the osteoporosis and related fracture. Alternatively, the combination and sequential therapies with anti-osteoporosis drugs are promising approaches for prevention and treatment of osteoporosis and have far-reaching significance.

Nanoparticles for the treatment of osteoporosis

Osteoporosis is by far the most frequent metabolic disease affecting bone. Current clinical therapeutic treatments are not able to offer long-term solutions. Most of the clinically used anti-osteoporotic drugs are administered systemically, which might lead to side effects in non-skeletal tissues. Therefore, to solve these disadvantages, researchers have turned to nanotechnologies and nanomaterials to create innovative and alternative treatments. One of the innovative approaches to enhance osteoporosis therapy and prevent potential adverse effects is the development of bone-targeting drug delivery technologies. It minimizes the systemic toxicity and also improves the pharmacokinetic profile and therapeutic efficacy of chemical drugs. This paper reviews the current available bone targeting drug delivery systems, focusing on nanoparticles, proposed for osteoporosis treatment. Bone targeting delivery systems is still in its infancy, thus, challenges are ahead of us, including the stability and the toxicity issues. Newly developed biomaterials and technologies with potential for safer and more effective drug delivery, require multidisciplinary collaboration between scientists from many different areas, such as chemistry, biology, engineering, medicine, etc, in order to facilitate their clinical applications.

The sensitivity of the DNA damage checkpoint prevents oocyte maturation in endometriosis.

Mouse oocytes respond to DNA damage by arresting in meiosis I through activity of the Spindle Assembly Checkpoint (SAC) and DNA Damage Response (DDR) pathways. It is currently not known if DNA damage is the primary trigger for arrest, or if the pathway is sensitive to levels of DNA damage experienced physiologically. Here, using follicular fluid from patients with the disease endometriosis, which affects 10% of women and is associated with reduced fertility, we find raised levels of Reactive Oxygen Species (ROS), which generate DNA damage and turn on the DDR-SAC pathway. Only follicular fluid from patients with endometriosis, and not controls, produced ROS and damaged DNA in the oocyte. This activated ATM kinase, leading to SAC mediated metaphase I arrest. Completion of meiosis I could be restored by ROS scavengers, showing this is the primary trigger for arrest and offering a novel clinical therapeutic treatment. This study establishes a clinical relevance to the DDR induced SAC in oocytes. It helps explain how oocytes respond to a highly prevalent human disease and the reduced fertility associated with endometriosis.

Advances in Nanotechnology for the Treatment of Osteoporosis.

Osteoporosis is a degenerative bone disease commonly related to aging. With an increase in life expectancies worldwide, the prevalence of the disease is expected to rise. Current clinical therapeutic treatments are not able to offer long-term solutions to counter the bone mass loss and the increased risk of fractures, which are the primary characteristics of the disease. However, the combination of bioactive nanomaterials within a biomaterial scaffold shows promise for the development of a localized, long-term treatment for those affected by osteoporosis. This review summarizes the unique characteristics of engineered nanoparticles that render them applicable for bone regeneration and recaps the current body of knowledge on nanomaterials with potential for osteoporosis treatment and bone regeneration. Specifically, we highlight new developments that are shaping this emerging field and evaluate applications of recently developed nanomaterials for osteoporosis treatment. Finally, we will identify promising new research directions in nanotechnology for bone regeneration.

Neuronal stem cells in the central nervous system and in human diseases

The process of cortical expansion in the central nervous system is a key step of mammalian brain development to ensure its physiological function. Radial glial (RG) cells are a glial cell type contributing to this progress as intermediate neural progenitor cells responsible for an increase in the number of cortical neurons. In this review, we discuss the current understanding of RG cells during neurogenesis and provide further information on the mechanisms of neurodevelopmental diseases and stem cell-related brain tumorigenesis. Knowledge of neuronal stem cell and relative diseases will bridge benchmark research through translational studies to clinical therapeutic treatments of these diseases.

Substance P receptor activation induces downregulation of the AMPA receptor functionality in cortical neurons from a genetic model of Amyotrophic Lateral Sclerosis

Substance P (SP), a neuropeptide member of the tachykinin (TK) family, has a functional role both in physiological and pathological conditions, including Amyotrophic Lateral Sclerosis disease. One hypothesis of the selective motor neuron death in ALS involves the excitatory neurotransmitter glutamate, because these neurons are extremely susceptible to excessive stimulation of AMPA receptors. It has been reported that SP exerts its action against a variety of insults including excitotoxicity, and that altered levels of SP have been observed in the cerebrospinal fluid (CSF) of patients with ALS. Here we have analyzed the interaction between SP and AMPA receptor functionality, both in Control cortical neurons in culture and in those obtained from a genetic mouse model of ALS (G93A). Our studies demonstrate that SP reduces the kainate-activated currents in Control and G93A neurons and that this reduction is significantly higher in the mutated neurons. SP effect is mediated by its receptor NK1 because GR 82334 (5 μM), a NK1 competitive antagonist, is able to suppress the current reduction. Analysis of miniature excitatory postsynaptic currents (mEPSCs) in Control and G93A neurons indicates that SP (200 nM) is able to significantly decrease the mEPSC amplitudes in G93A neurons, whereas it is ineffective on Control mEPSCs. Western blotting experiments in cultures and cortical tissues show a higher NK1 expression level in G93A mice compared to that of Control. This is also confirmed by immunocytochemistry experiments in cultured neurons. In addition, the amount of GluR1 subunit AMPA receptors is not modified following SP exposure, indicating a non internalization of the AMPA receptors. Finally, toxicity experiments have revealed that SP is able to rescue G93A cortical cells whereas it is ineffective on those of Control. These findings provide the first evidence of SP having a physiological and protective role in the G93A mouse model of ALS, and may suggest the possible use of SP as a clinical therapeutic treatment.

Polyacrylate nanoparticles: toxicity or new nanomedicine?

To the Editors: Nanotoxicology is now becoming a very important field in view of the unique physicochemical properties of nanomaterials that may lead to adverse biological effects on occupational, environmental and consumer matters. However, a conclusion on the toxicity of nanomaterials should be carefully drawn taking into account the complexity of determining the toxicity of nanoscale materials. Until now, studies on the adverse outcomes of nanomaterials, such as carbon nanotubes and nanoparticles, were limited to the experimental stage. Although there were some reports on human cell lines, the research was limited to in vitro studies; in vivo studies were completely limited to animals. Very recently, researchers from Beijing Chaoyang Hospital, Beijing, China, have reported the first case of the clinical toxicity of polyacrylate nanoparticles in humans 1. The main evidence involves pathological examination of patients’ lung tissue and observations by transmission electron microscopy. Nonspecific pulmonary inflammation, pulmonary fibrosis and foreign-body granulomas of pleura were displayed. Round nanoparticles ∼30 nm in diameter were observed to lodge in the cytoplasm and karyoplasm of pulmonary epithelial and mesothelial cells and also in chest fluid. Based on these clinical observations and analysis, the authors conclude that long-term exposure to polyacrylate nanoparticles could result in serious damage to human lungs. Here, we note that their work may provide important evidence for concerns about the toxicity of nanoparticles in humans and may afford promising opportunities to develop a new clinical therapeutic treatment for such nanoparticle-based diseases. However, we would like to point out that there are not enough data to strongly suggest that the clinical symptoms of the patients result from the toxicity of polyacrylate nanoparticles themselves. Therefore, adequate experimental evidence is very necessary for supporting the claimed toxicity, such as in vivo and in vitro toxicological assessments of polyacrylate nanoparticles, control experiments using animals …

Profili di sensibilità agli agenti antifungini di lieviti e miceti lievito-simili isolati negli ospedali torinesi

There is an increased incidence of the morbidity and mortality associated with fungal infections caused by resistant fungi in various groups of patients; therefore the monitoring of their susceptibility to antifungal agents to be necessary for optimizing clinical therapeutic treatment. The susceptibility profiles of recent clinical yeasts and yeast-like fungi isolated from Turin’s Hospitals to current antifungal drugs are here reported. Candida albicans was the most frequently isolated species (40%), followed by C. glabrata (22%).A high rate of susceptibility to all antifungal agents tested was observed and low evidence of resistance was found.