Fast Ultrasound Scanning is a Rapid, Sensitive, Precise and Cost-Effective Method to Monitor Tumor Grafts in Mice.

In preclinical studies, accurate monitoring of tumor dynamics is crucial for understanding cancer biology and evaluating therapeutic interventions. Traditional methods like caliper measurements and bioluminescence imaging (BLI) have limitations, prompting the need for improved imaging techniques. This study introduces a fast-scan high-frequency ultrasound (HFUS) protocol for the longitudinal assessment of syngeneic breast tumor grafts in mice, comparing its performance with caliper, BLI measurements and with histological analysis. The E0771 mammary gland tumor cell line, engineered to express luciferase, was orthotopically grafted into immunocompetent C57BL/6 mice. Tumor growth was monitored longitudinally at multiple timepoints using caliper measurement, HFUS, and BLI, with the latter two modalities assessed against histopathological standards post-euthanasia. The HFUS protocol was designed for rapid, anesthesia-free scanning, focusing on volume estimation, echogenicity, and necrosis visualization. All mice developed tumors, only 20.6% were palpable at day 4. HFUS detected tumors as small as 2.2 mm in average diameter from day 4 post-implantation, with an average scanning duration of 47 s per mouse. It provided a more accurate volume assessment than caliper, with a lower average bias relative to reference tumor volume. HFUS also revealed tumor necrosis, correlating strongly with BLI in terms of tumor volume and cellularity. Notable discrepancies between HFUS and BLI growth rates were attributed to immune cell infiltration. The fast HFUS protocol enables precise and efficient tumor assessment in preclinical studies, offering significant advantages over traditional methods in terms of speed, accuracy, and animal welfare, aligning with the 3R principle in animal research.

Metabolomic Impact of Lidocaine on a Triple Negative Breast Cancer Cell Line.

Background: Metabolomics and onco-anesthesia are two emerging research fields in oncology. Metabolomics (metabolites analysis) is a new diagnostic and prognostic tool that can also be used for predicting the therapeutic or toxic responses to anticancer treatments. Onco-anesthesia studies assess the impact of anesthesia on disease-free and overall survival after cancer surgery. It has been shown that local anesthetics (LA), particularly lidocaine (LIDO), exert antitumor properties both in vitro and in vivo and may alter the biologic fingerprints of cancer cells. As LA are known to impair mitochondrial bioenergetics and byproducts, the aim of the present study was to assess the impact of LIDO on metabolomic profile of a breast cancer cell line. Methods: Breast cancer MDA-MB-231 cells were exposed for 4 h to 0.5 mM LIDO or vehicle (n = 4). The metabolomic fingerprint was characterized by high resolution magic angle spinning NMR spectroscopy (HRMAS). The multivariate technique using the Algorithm to Determine Expected Metabolite Level Alteration (ADEMA) (Cicek et al., PLoS Comput. Biol., 2013, 9, e1002859), based on mutual information to identify expected metabolite level changes with respect to a specific condition, was used to determine the metabolites variations caused by LIDO. Results: LIDO modulates cell metabolites levels. Several pathways, including glutaminolysis, choline, phosphocholine and total choline syntheses were significantly downregulated in the LIDO group. Discussion: This is the first study assessing the impact of LIDO on metabolomic fingerprint of breast cancer cells. Among pathways downregulated by LIDO, many metabolites are reported to be associated with adverse prognosis when present at a high titer in breast cancer patients. These results fit with the antitumor properties of LIDO and suggest its impact on metabolomics profile of cancer cells. These effects of LIDO are of clinical significance because it is widely used for local anesthesia with cutaneous infiltration during percutaneous tumor biopsy. Future in vitro and preclinical studies are necessary to assess whether metabolomics analysis requires modification of local anesthetic techniques during tumor biopsy.

Identification of driver genes for critical forms of COVID-19 in a deeply phenotyped young patient cohort.

The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 "critical" (in the intensive care unit under mechanical ventilation) and 25 "non-critical" (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease ADAM9. This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified ADAM9 as a driver of disease severity and a candidate therapeutic target.

Detection of Pneumocystis jirovecii in Patients with Severe COVID-19: Diagnostic and Therapeutic Challenges.

Cases of Pneumocystis jirovecii pneumonia (PCP) in patients suffering from COVID-19 were described in patients with various comorbidities and outcomes. The diagnosis of PCP in these patients is difficult due to clinical and radiological similarities. We carried out this study in order to better describe potentially at-risk patients and their outcomes. We retrospectively analyzed all patients with a P. jirovecii PCR performed in bronchoalveolar lavage fluid, tracheal aspirate, or sputum within a month after the COVID-19 diagnosis. Fifty-seven patients with COVID-19 infection were tested for P. jirovecii. Among 57 patients with COVID-19, four patients had a concomitant positive P. jirovecii PCR. These four patients were elderly with a mean age of 78. Two patients were immunocompromised, and the two others presented only diabetes mellitus. Three patients presented an ARDS requiring transfer to the ICU and mechanical ventilation. All patients presented lymphocytopenia. Three patients had probable PCP, and one had proven PCP. All patients died within two months after hospital admission. These co-infections are rare but severe, therefore, PCP should be considered in case of worsening of the condition of patients with severe COVID-19.

Impact of intravenous lidocaine on clinical outcomes of patients with ARDS during COVID-19 pandemia (LidoCovid): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The main objective of this study is to evaluate the effect of intravenous lidocaine on gas exchange and inflammation in acute respiratory distress syndrome due or not to Covid-19 pneumonia. TRIAL DESIGN: This is a prospective monocentric, randomized, quadruple-blinded and placebo-controlled superiority trial. This phase 3 clinical study is based on two parallel groups received either intravenous lidocaine 2% or intravenous NaCl 0.9%. PARTICIPANTS: This study has been conducted at the University Hospitals of Strasbourg (medical and surgical Intensive Care Units in Hautepierre Hospital) since the 4th November 2020. The participants are 18 years-old and older, hospitalized in ICU for a moderate to severe ARDS according to the Berlin definition; they have to be intubated and sedated for mechanical protective ventilation. All participants are affiliated to the French Social security system and a dosage of beta HCG has to be negative for women of child bearing age . For the Covid-19 subgroup, the SARS-CoV2 infection is proved by RT-PCR <7 days before admission and/or another approved diagnostic technique and/or typical CT appearance pneumonia. The data are prospectively collected in e-Case Report Forms and extracted from clinical files. INTERVENTION AND COMPARATOR: The participants are randomised in two parallel groups with a 1:1 ratio. In the experimental group, patients receive intravenous lidocaine 2% (20mg/mL) (from FRESENIUS KABI France); the infusion protocol provide a bolus of 1 mg/kg (ideal weight), followed by 3 mg/kg/h for the first hour, 1.5 mg/kg/h for the second hour, 0.72 mg/kg/h for the next 22 hours and then 0.6 mg/kg/h for 14 days at most or 24 hours after extubation or ventilator-weaning. The patients in the control group receive intravenous NaCl 0.9% (9 mg/mL) (from Aguettant, France) as placebo comparator; the infusion protocol provide a bolus of 0.05 mL/kg (ideal weight), followed by 0.15 mL/kg/h for the first hour, 0.075 mL/kg/h for the second hour, 0.036 mL/kg/h for the next 22 hours, and the 0.03 mL/kg/h for up to 14 days or 24 hours after extubation or ventilator-weaning. Lidocaine level is assessed at H4, D2, D7 and D14 to prevent local anesthetics systemic toxicity. Clinical data and biological samples are collected to assess disease progression. MAIN OUTCOMES: The primary outcome is the evolution of alveolar-capillary gas exchange measured by the PaO2/FiO2 ratio after two days of treatment. The secondary endpoints of the study include the following: Evolution of PaO2/FiO2 ratio at admission and after 21 days of treatment Number of ventilator-free days Anti-inflammatory effects by dosing inflammatory markers at different timepoints (ferritin, bicarbonate, CRP, PCT, LDH, IL-6, Troponin HS, triglycerides, complete blood count, lymphocytes) Anti-thrombotic effects by dosing platelets, aPTT, fibrinogen, D-dimers, viscoelastic testing and identification of all thromboembolic events up to 4 weeks. Plasmatic concentration of lidocaine and albumin Incidence of adverse events like cardiac rhythm disorders, need of vasopressors, any modification of the QRS, QTc or PR intervals every day Ileus recovery time Consumption of hypnotics, opioids, neuromuscular blockers. Lengths of stay in the ICU, incidence of reintubation and complications due to intensive care unit care (mortality until 90 days, pneumothorax, bacterial pneumopathy, bronchospasm, cardiogenic shock, acute renal failure, need of renal dialysis, delirium, atrial fibrillation, stroke (CAM-ICU score), tetraplegia (MCR score)). Incidence of cough and sore throat at extubation or ventilator-weaning and within 24 hours. All these outcomes will be evaluated according to positivity to Sars-Cov-2. RANDOMISATION: The participants who meet the inclusion criteria and have signed written informed consent will be randomly allocated using a computer-generated random number to either intervention group or control group. The distribution ratio of the two groups will be 1:1, with a stratification according to positivity to Sars-Cov-2. BLINDING (MASKING): All participants, care providers, investigator and outcomes assessor are blinded. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): We planned to randomize fifty participants in each group, 100 participants total. TRIAL STATUS: The amended protocol version 2.1 was approved by the Ethics Committee "Comité de Protection des Personnes Sud-Méditerranée II on January 8, 2021 and by the Commission Nationale de l'Informatique et des Libertés (CNIL) on November 10, 2020. The study is currently recruiting participants; the recruitment started in November 2020 and the planned recruitment period is three years. TRIAL REGISTRATION: The trial was registered on clinicaltrials.gov on October 30, 2020 and identified by number NCT04609865 . FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Antitumor Effects of Lidocaine on Human Breast Cancer Cells: An In Vitro and In Vivo Experimental Trial.

AIM: Retrospective studies have suggested a protective effect of regional anesthesia against recurrence after cancer surgery. But confirmation of the in vivo antitumor effects is lacking. We examined the in vitro antitumor effects of lidocaine on various breast cancer cell lines and then assessed these properties in vivo at clinically relevant concentrations. MATERIALS AND METHODS: In vitro experiments: normal breast epithelial cells (NBEC) MCF-10A and three tumor breast epithelial cells (TBEC) lines (MCF-7 luminal A, MDA-MB-231 triple-negative and SKBr3 HER2 positive) were exposed to increasing concentrations of lidocaine. Cell viability, migration and anchorage-independent growth were assessed by MTT, wound healing, and soft-agar growth assays. In vivo experiments: 6-week-old severe combined immunodeficient mice were injected intraperitoneally with MDA-MB-231 cells and were treated with intraperitoneal lidocaine or phosphate-buffered saline. The mice were euthanized when they reached experimental endpoints or sacrificed to determine peritoneal carcinomatosis index and global tumor volumes. RESULTS: Lidocaine reduced the viability of all the cell lines, inhibited migration of TBEC compared to the NBEC, and compromised the anchorage-independent growth of the triple-negative cells. Intraperitoneal lidocaine improved survival of mice with MDA-MB-231 peritoneal carcinomatosis using doses that are consistent with the current clinical settings for analgesia. CONCLUSION: In agreement with the notion that local anesthesia may be beneficial for cancer therapy, lidocaine has a protective effect against breast cancer cells in experimental studies. However, the beneficial impact of local anesthetics on breast cancer needs to be strengthened by additional preclinical and clinical trials.

Skeletal Muscle and Lymphocyte Mitochondrial Dysfunctions in Septic Shock Trigger ICU-Acquired Weakness and Sepsis-Induced Immunoparalysis.

Fundamental events driving the pathological processes of septic shock-induced multiorgan failure (MOF) at the cellular and subcellular levels remain debated. Emerging data implicate mitochondrial dysfunction as a critical factor in the pathogenesis of sepsis-associated MOF. If macrocirculatory and microcirculatory dysfunctions undoubtedly participate in organ dysfunction at the early stage of septic shock, an intrinsic bioenergetic failure, sometimes called "cytopathic hypoxia," perpetuates cellular dysfunction. Short-term failure of vital organs immediately threatens patient survival but long-term recovery is also severely hindered by persistent dysfunction of organs traditionally described as nonvital, such as skeletal muscle and peripheral blood mononuclear cells (PBMCs). In this review, we will stress how and why a persistent mitochondrial dysfunction in skeletal muscles and PBMC could impair survival in patients who overcome the first acute phase of their septic episode. First, muscle wasting protracts weaning from mechanical ventilation, increases the risk of mechanical ventilator-associated pneumonia, and creates a state of ICU-acquired muscle weakness, compelling the patient to bed. Second, failure of the immune system ("immunoparalysis") translates into its inability to clear infectious foci and predisposes the patient to recurrent nosocomial infections. We will finally emphasize how mitochondrial-targeted therapies could represent a realistic strategy to promote long-term recovery after sepsis.

The Amide Local Anesthetic Lidocaine in Cancer Surgery-Potential Antimetastatic Effects and Preservation of Immune Cell Function? A Narrative Review.

Surgical removal of the primary tumor in solid cancer is an essential component of the treatment. However, the perioperative period can paradoxically lead to an increased risk of cancer recurrence. A bimodal dynamics for early-stage breast cancer recurrence suggests a tumor dormancy-based model with a mastectomy-driven acceleration of the metastatic process and a crucial role of the immunosuppressive state during the perioperative period. Recent evidence suggests that anesthesia could also influence the progress of the disease. Local anesthetics (LAs) have long been used for their properties to block nociceptive input. They also exert anti-inflammatory capacities by modulating the liberation or signal propagation of inflammatory mediators. Interestingly, LAs can reduce viability and proliferation of many cancer cells in vitro as well. Additionally, retrospective clinical trials have suggested that regional anesthesia for cancer surgery (either with or without general anesthesia) might reduce the risk of recurrence. Lidocaine, a LA, which can be administered intravenously, is widely used in clinical practice for multimodal analgesia. It is associated with a morphine-sparing effect, reduced pain scores, and in major surgery probably also with a reduced incidence of postoperative ileus and length of hospital stay. Systemic delivery might therefore be efficient to target residual disease or reach cells able to form micrometastasis. Moreover, an in vitro study has shown that lidocaine could enhance the activity of natural killer (NK) cells. Due to their ability to recognize and kill tumor cells without the requirement of prior antigen exposure, NKs are the main actor of the innate immune system. However, several perioperative factors can reduce NK activity, such as stress, pain, opioids, or general anesthetics. Intravenous lidocaine as part of the perioperative anesthesia regimen would be of major interest for clinicians, as it might bear the potential to reduce the risk of cancer recurrence or progression patients undergoing cancer surgery. As a well-known pharmaceutical agent, lidocaine might therefore be a promising candidate for oncological drug repurposing. We urgently need clinical randomized trials assessing the protective effect of lidocaine on NKs function and against recurrence after cancer surgery to achieve a "proof of concept."

Skeletal muscle ischemia-reperfusion injury and cyclosporine A in the aging rat.

Old patients exhibit muscle impairments and increased perioperative risk during vascular surgery procedures. Although aging generally impairs protective mechanisms, data are lacking concerning skeletal muscle in elderly. We tested whether cyclosporine A (CsA), which protects skeletal muscle from ischemia-reperfusion (IR) in young rats, might reduce skeletal muscle mitochondrial dysfunction and oxidative stress in aging rats submitted to hindlimb IR. Wistar rats aged 71-73 weeks were randomized to IR (3 h unilateral tourniquet application and 2 h reperfusion) or IR + CsA (10 mg/kg cyclosporine IV before reperfusion). Maximal oxidative capacity (VM ax ), acceptor control ratio (ACR), and relative contribution of the mitochondrial respiratory chain complexes II, III, IV (VS ucc ), and IV (VTMPD /Asc ), together with calcium retention capacity (CRC) a marker of apoptosis, and tissue reactive oxygen species (ROS) production were determined in gastrocnemius muscles from both hindlimbs. Compared to the nonischemic hindlimb, IR significantly reduced mitochondrial coupling, VMax (from 7.34 ± 1.50 to 2.87 ± 1.22 µMO2 /min/g; P < 0.05; -70%), and VS ucc (from 6.14 ± 1.07 to 3.82 ± 0.83 µMO2 /min/g; P < 0.05; -42%) but not VTMPD /Asc . IR also decreased the CRC from 15.58 ± 3.85 to 6.19 ± 0.86 µMCa(2+) /min/g; P < 0.05; -42%). These alterations were not corrected by CsA (-77%, -49%, and -32% after IR for VM ax, VS ucc , and CRC, respectively). Further, CsA significantly increased ROS production in both hindlimbs (P < 0.05; +73%). In old rats, hindlimb IR impairs skeletal muscle mitochondrial function and increases oxidative stress. Cyclosporine A did not show protective effects.

Oxidative stress precedes skeletal muscle mitochondrial dysfunction during experimental aortic cross-clamping but is not associated with early lung, heart, brain, liver, or kidney mitochondrial impairment.

OBJECTIVE: Lower limb ischemia-reperfusion results in skeletal muscle mitochondrial alterations, production of reactive oxygen species (ROS), and remote organ impairments that are largely involved in patient prognosis. However, whether ischemia without reperfusion increases ROS production and precedes mitochondrial alteration and whether mitochondrial dysfunction occurs early in remote organs is unknown. This study determined muscle mitochondrial function and ROS production after ischemia alone, or followed by two periods of reperfusion, and investigated heart, lung, liver, kidney, and brain mitochondrial functions after lower limb ischemia-reperfusion. METHODS: Wistar rats were randomized into four groups: sham (aortic exposure but no ischemia, n = 9), I3 (ischemia alone induced by aortic cross-clamping for 3 hours, n = 9), I3R10' and I3R2 (aortic cross-clamping, followed by reperfusion for 10 minutes [n = 8] or 2 hours [n = 9]). Blood lactate, alanine aminotransferase, aspartate aminotransferase, and creatinine were measured. Mitochondrial respiratory chain complexes I, II, III, and IV activities and mitochondrial coupling (acceptor control ratio) were analyzed using a Clark oxygen electrode in skeletal muscle, lung, heart, brain, liver, and kidney. ROS production was determined using dihydroethidium staining in muscle, heart, liver, and kidney. Inflammation was also investigated in remote organs (heart, liver, and kidney) using monocyte-macrophage-2 antibody staining. RESULTS: Lactate level increased after ischemia in all groups. In muscle, ROS increased significantly after ischemia alone (+324% ± 66%; P = .038), normalized after 10 minutes of reperfusion, and increased again at 2 hours of reperfusion (+349.2 ± 67%; P = .024). Interestingly, mitochondrial function was unaffected by ischemia alone or followed by 10 minutes of reperfusion, but maximal mitochondrial oxidative capacity (6.10 ± 0.51 vs. 4.24 ± 0.36 µmol/min/g, -30%; P < .05) and mitochondrial coupling decreased after 2 hours of reperfusion (1.93 ± 0.17 vs. 1.33 ± 0.07, -45%; P < .01), in sham and I3R2 rats, respectively. Despite increased serum aspartate aminotransferase (×13; P < .0001), alanine aminotransferase (×6; P = .0019), and creatinine (×3; P = .0004), remote organs did not show mitochondrial alteration, inflammation, or ROS production enhancement after 2 hours of reperfusion. CONCLUSIONS: Oxidative stress precedes skeletal muscle mitochondrial dysfunction during lower limb ischemia. Such a kinetic explains the efficacy of ischemic preconditioning and supports that therapy should be conducted even during ongoing ischemia, suggesting that ischemic preconditioning might be a successful approach.