Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

Cochlear Microphonics in Hearing Preservation Cochlear Implantees.

OBJECTIVES: The intracochlear electrocochleography (ECoG) could be recorded directly from the cochlear implant (CI) electrode in CI recipients with residual hearing. The primary objective of this study is to identify the most sensitive frequency to record cochlear microphonics (CM) in CI users with a wide degree of hearing abilities and deep electrode insertion. The secondary objective is to identify the optimum location within the cochlea to record intracochlear potentials. MATERIALS AND METHODS: CMs were recorded from the CI electrodes in eight females and eight males implanted with CIs Pulsar, Concerto, or Sonata, Med-El Corp. RESULTS: Among the tone pips of various frequencies, 1k or 500 Hz were the most sensitive for CI users. The most sensitive place in the cochlea to record the CM potentials depended on the tone frequency used. The deeper into the cochlea the mean maximum CM peak-to-peak amplitude was measured, the lower the stimulating tone frequency was. CONCLUSION: The most optimal recording parameters identified for intracochlear CM recording can be useful for intraoperative and postoperative monitoring of cochlear health in CI users with residual hearing.

Cysteamine, an Endogenous Aminothiol, and Cystamine, the Disulfide Product of Oxidation, Increase Pseudomonas aeruginosa Sensitivity to Reactive Oxygen and Nitrogen Species and Potentiate Therapeutic Antibiotics against Bacterial Infection.

Cysteamine is an endogenous aminothiol produced in mammalian cells as a consequence of coenzyme A metabolism through the activity of the vanin family of pantetheinase ectoenzymes. It is known to have a biological role in oxidative stress, inflammation, and cell migration. There have been several reports demonstrating anti-infective properties targeting viruses, bacteria, and even the malarial parasite. We and others have previously described broad-spectrum antimicrobial and antibiofilm activities of cysteamine. Here, we go further to demonstrate redox-dependent mechanisms of action for the compound and how its antimicrobial effects are, at least in part, due to undermining bacterial defenses against oxidative and nitrosative challenges. We demonstrate the therapeutic potentiation of antibiotic therapy against Pseudomonas aeruginosa in mouse models of infection. We also demonstrate potentiation of many different classes of antibiotics against a selection of priority antibiotic-resistant pathogens, including colistin (often considered an antibiotic of last resort), and we discuss how this endogenous antimicrobial component of innate immunity has a role in infectious disease that is beginning to be explored and is not yet fully understood.

Effects of bariatric surgery on cardiovascular risk factors among morbidly obese patients.

AIM OF THE STUDY: The aim of this study was to evaluate the influence of laparoscopic sleeve gastrectomy and laparoscopic Roux-en-Y gastric by-pass on risk factors of cardiovascular diseases. MATERIAL AND METHODS: We analyzed prospectively collected data of patients operated for morbid obesity who were qualified for laparoscopic sleeve gastrectomy (LSG) or laparoscopic Roux-en-Y gastric by-pass (LRYGB). Risk factors for wylaczecardiovascular diseases were assessed with the SCORE scale and both full and hard Framingham cardiovascular risk scores (FCRs). The data were collected on admission and one year after the procedures. We enrolled 264 patients (119 females, 116 males, 40.2±9.9 years old), of whom 117 underwent LRYGB and 118 LSG, respectively. RESULTS: Preoperatively, 12% of patients were in the high-risk category of the SCORE scale, 65% were in the moderate risk category, and 24% were in the low-risk category. The median score of the SCORE scale was 1 (1-2). Lipid-based full FCR was 34.5% (24%-68%) and the hard FCR was 17.5% (10%-52%), while the respective BMI-based FCRs were 59% (31%-84%) and 37% (15%-67%). One year after the procedures, the mean %EBMIL (62.88%±20.02%) and %EWL (53.18%±15.87) were comparable between both procedures. Hypertension treatment was not necessary in 33 patients after LSG and in 55 after LRYGB. Diabetes mellitus remitted in 9 and 29 patients, respectively. Both procedures significantly reduced high and moderate risk prevalence in the SCORE scale in favor of the low risk category. Surgical interventions resulted in significant reductions of FCRs 1 year after surgery ( p<0.001). CONCLUSIONS: Both LSG and LRYGB lead to a significant and comparable body mass reduction. Both procedures significantly decrease of the risk of cardiovascular diseases, based on SCORE and Framingham scales.

[Metformin and changes in blood pressure and heart rate in lean patients with polycystic ovary syndrome (PCOS)--preliminary study]. / Metformina, a zmiany cisnienia tetniczego krwi i czestosci akcji serca u szczuplych pacjentek z zespolem policystycznych jajników (PCOS)--doniesienie wstepne.

INTRODUCTION: The aim of our study was to assess the value of blood pressure and heart rate using the 24-hour blood pressure monitoring (ABPM) before and after treatment with metformin to patients with polycystic ovary syndrome (PCOS) and normal lean. MATERIAL AND METHODS: 5 patients received metformin 1500 mg per day in three divided doses. ABPM was performed to each patient with PCOS twice: before and after 6 months of treatment with metformin. RESULTS: In patients with PCOS and normal lean after treatment with metformin we observed: statistically significant lower systolic blood pressure (120.2 ± 22.33 mmHg vs 113.22 ± 21.43 mm Hg, p = 0.0248); lower systolic blood pressure of daily measurements (127.1 ± 32.13 mmHg vs 116.1 ± 22.08 mmHg, p = 0.0062); reduction in average arterial pressure MAP in the measurement of the day (95.52 ± 22.76 mmHg vs 88.36 ± 16.41 mmHg, p = 0.048); oscillometric pressure reduction (96.27 ± 27.93 mmHg vs 87.82 ± 21.61, p = 0.0004 mmHg); oscillometric pressure reduction of daily measurements (102.1 ± 27.93 mmHg vs 91.85 ± 21.61 mmHg, p = 0.0032); oscillometric pressure reduction in the measure- ment of the night (88.81 ± 24.85 mmHg vs 82.22 ± 20.54 mmHg, p = 0.0089). In women after treatment with metformin has also been observed higher average heart rate (65.82 ± 13.48 / min vs. 70.71 ± 16.04 min; p < 0.01). The calculations included 500 measurements. CONCLUSION: Treatment with metformin in patients with PCOS and normal lean leads to lower blood pressure and increases the frequency of heart rate.

Cysteamine (Lynovex®), a novel mucoactive antimicrobial & antibiofilm agent for the treatment of cystic fibrosis.

BACKGROUND: There remains a critical need for more effective, safe, long-term treatments for cystic fibrosis (CF). Any successful therapeutic strategy designed to combat the respiratory pathology of this condition must address the altered lung physiology and recurrent, complex, polymicrobial infections and biofilms that affect the CF pulmonary tract. Cysteamine is a potential solution to these unmet medical needs and is described here for the first time as (Lynovex®) a single therapy with the potential to deliver mucoactive, antibiofilm and antibacterial properties; both in oral and inhaled delivery modes. Cysteamine is already established in clinical practice for an unrelated orphan condition, cystinosis, and is therefore being repurposed (in oral form) for cystic fibrosis from a platform of over twenty years of safety data and clinical experience. METHODS: The antibacterial and antibiofilm attributes of cysteamine were determined against type strain and clinical isolates of CF relevant pathogens using CLSI standard and adapted microbiological methods and a BioFlux microfluidic system. Assays were performed in standard nutrient media conditions, minimal media, to mimic the low metabolic activity of microbes/persister cells in the CF respiratory tract and in artificial sputum medium. In vivo antibacterial activity was determined in acute murine lung infection/cysteamine nebulisation models. The mucolytic potential of cysteamine was assessed against DNA and mucin in vitro by semi-quantitative macro-rheology. In all cases, the 'gold standard' therapeutic agents were employed as control/comparator compounds against which the efficacy of cysteamine was compared. RESULTS: Cysteamine demonstrated at least comparable mucolytic activity to currently available mucoactive agents. Cysteamine was rapidly bactericidal against both metabolically active and persister cells of Pseudomonas aeruginosa and also emerging CF pathogens; its activity was not sensitive to high ionic concentrations characteristic of the CF lung. Cysteamine prevented the formation of, and disrupted established P. aeruginosa biofilms. Cysteamine was synergistic with conventional CF antibiotics; reversing antibiotic resistance/insensitivity in CF bacterial pathogens. CONCLUSIONS: The novel mucolytic-antimicrobial activity of cysteamine (Lynovex®) provides potential for a much needed new therapeutic strategy in cystic fibrosis. The data we present here provides a platform for cysteamine's continued investigation as a novel treatment for this poorly served orphan disease.