Demonstrating the Manufacture of Human CAR‐T Cells in an Automated Stirred‐Tank Bioreactor

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 10⁶ cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.     

Vitreoscilla hemoglobin renders Enterobacter aerogenes highly susceptible to heavy metals

When expressed in heterologous microorganisms Vitreoscilla hemoglobin (VHb) acts as oxygen storage and causes a higher oxygen uptake. In this study, the effect of this protein on growth, sensitivity and antioxidant properties of Enterobacter aerogenes exposed to metal stress was investigated. The strain expressing VHb was more sensitive to mercury and cadmium as the minimal inhibitory concentration (MIC) for these metals was up to 2-fold lower in this strain than the host and the recombinant strain carrying a comparable plasmid. At lower concentrations than MIC, the metals partially limited growth and caused an inhibition proportional to metal concentration applied. The growth pattern of VHb expressing strain was also distinctly different from other two non-hemoglobin strains. The hemoglobin containing strain showed substantially higher superoxide dismuates (SOD) activity than the non-hemoglobin strains, while catalase levels were similar in all strains. All strains exposed to copper, however, showed similar MIC values, growth patterns, and SOD and catalase levels.     

Antimicrobial peptides in the duodenum at the acute and convalescent stages in patients with diarrhea due to Vibrio cholerae O1 or enterotoxigenic Escherichia coli infection

Patients with acute watery diarrhea caused by Vibrio cholerae O1 or enterotoxigenic Escherichia coli (ETEC) were analyzed for innate immune factors produced by the epithelium during the disease process. Duodenal biopsies were obtained from study participants at the acute (day 2) and convalescent (day 21) stages of disease. Levels of α-defensin (HD-5 and -6), β-defensin (hBD-1-4), and cathelicidin (LL-37) mRNAs were determined by real-time qRT-PCR. hBD-2, HD-5, LL-37 peptides were analyzed in duodenal epithelium by immunomorphometry. Concentration of hBD-2 in stool was determined by ELISA. Specimens from healthy controls were also analyzed. hBD-2 mRNA levels were significantly increased at acute stage of diarrhea; hBD-2 peptide was detected in fecal specimens but barely in duodenal epithelium at acute stage. Immunomorphometry analysis showed that Paneth cells contain significantly higher amounts of HD-5 pre/propeptide at convalescence (P<0.01) and in healthy controls (P<0.001) compared to acute stage, LL-37 peptide levels also decreased at acute stage while mRNA levels remained unchanged. mRNA expression levels of the other antimicrobial peptides remained unchanged with higher levels of α-defensins than β-defensins. V. cholerae induced an innate immune response at the acute stage of disease characterized by increased expression of hBD-2, and continued expression of hBD-1, HD-5-6, and LL-37.     

PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo

The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.     

Biomarkers predicting tumor response and evasion to anti-angiogenic therapy

No fully validated biological markers currently exist to predict responsiveness to or the development of evasion to anti-angiogenic therapy of cancer. The identification of such biomarkers is vital to move these therapies forward, as failure to respond to these treatments is often associated with rapid tumor progression that could have been averted had the intrinsic or acquired evasion to anti-angiogenic therapy been identified in a timely fashion. Furthermore, the high cost of antiangiogenic therapies makes it important to avoid utilizing them in the setting of lack of response or developing evasion, making the identification of biomarkers even more important. A number of potential physiologic, circulating, tissue, and imaging biomarkers have emerged from recently completed preclinical animal studies and clinical trials. In this review, we define 5 different types of biomarkers (physiologic, circulating, intratumoral, genetic polymorphisms, and radiographic); discuss the challenges in establishing biomarkers of antiangiogenic therapy in animal models and in clinical trials; and discuss future strategies to identify and validate biomarkers of anti-angiogenic therapy.     

Wolff-Parkinson-White syndrome with an unroofed coronary sinus without persistent left superior vena cava treated with catheter cryoablation

Coronary sinus anomalies are rare congenital defects which are usually coexistent with a persistent left superior vena cava and may be associated with cardiac arrhythmias. We report an unroofed coronary sinus without persistent left superior vena cava diagnosed during a catheter ablation procedure for Wolff-Parkinson-White syndrome. Diagnostic and therapeutic options and outcomes are discussed. This condition is of relevance to electrophysiologists performing catheter-based procedures, as well as cardiologists implanting coronary sinus pacing leads, who may encounter this anomaly in their practice.     

Bacterial Spectrum and Antimicrobial Susceptibility Pattern of Bloodstream Infections in Children with Febrile Neutropenia: Experience of Single Center in Southeast of Turkey

Empirical antimicrobial therapy is usually started in febrile neutropenic patients without having culture results. The aim of this study was to help determine the policies of empirical antibiotic usage in febrile neutropenic children by detecting the antimicrobial susceptibility profile in this group of patients. In this study 811 blood cultures taken from neutropenic children hospitalized at the Department of Oncology of Gaziantep Children Hospital November 2007 and February 2010 were retrospectively evaluated. Blood cultures were routinely collected in aerobic and anaerobic media and incubated using the BACTEC system. Identification and antimicrobial susceptibility testing of the isolates to antimicrobial agents was performed using the Vitek2^® system according to the recommendations of the Clinical and Laboratory Standards Institute. Of 811 isolates analyzed, 128 (56.4%) were gram positive cocci, 43 (18.9%) were gram negative bacilli and fungi accounted for 56 (24.7%). The main isolated Gram-positive bacteria from blood were coagulase-negative staphylococcus (56.7%), followed by methicillin-resistant Staphylococcus aureus (14.1%). S. aureus and Streptococcus spp. were all susceptible to linezolid, vancomycin and teicoplanin. S aureus was still susceptible to few other antimicrobial agents such as tetracycline (82.4%), chloramphenicol (55.6%). Seven E. faecium, 7 E. fecalis and 1 E. hirae was isolated from blood cultures. Vancomycin resistance was detected in 6 out of 15 (40%) Enterococcus spp. isolates. Among gram-negative bacteria E. coli (30.2%) was followed by Klebsiella pneumoniae (20.9%) and Proteus spp. (18.6%). Imipenem (89.2%), meropenem (86.6%), chloramphenicol (88.9%), amicasin (82.4%) and fosfomycin (81.3%) showed highest susceptibility in vitro activity against all Gram-negative isolates. To know the antimicrobial susceptibility profile of the pathogens frequently isolated from febrile neutropenic children and to consider this profile before starting an empirical antibiotic therapy would help the clinics which have any role in the treatment of these patients to determine the empirical antibiotic usage policies.     

Morpho-Molecular Characterization of Soil Inhabitant Dermatophytes from Ahvaz,Southwest of Iran,a High Occurrence of <Emphasis Type="Italic">Microsporum fulvum</Emphasis>

Occurrence and diversity of dermatophyte mycoflora in 298 soil samples from Ahvaz, Southwest of Iran was investigated by using the hair-baiting technique. The samples were collected during spring (n = 210) and autumn (n = 88) of 2015, and the fungal isolates were identified based on the macro- and micro-morphology of colonies and with further ITS-rDNA RFLP and sequencing. Totally, 60 soil samples (20.1%) were positive for dermatophyte growth whose pH varied from 7.0 to 7.9. The highest (26.6%) and the lowest (14.3%) recovery rates were from the animal resorts and the streets soils samples, respectively. Seasonally, 16.7% of the spring samples and 28.4% of the autumn samples were positive. Based on molecular identification, three species of two genera were identified viz. M. fulvum (n = 57), M. canis (n = 2) and zoophilic Trichophyton interdigitale (n = 1). As a specific goal in the study, differentiation of the species in Microsporum gypseum complex was established by measuring the mean length and width of macroconidia in some strains of M. gypseum, M. fulvum and M. incurvatum. Mean size for macroconidia length and width in three species showed that M. gypseum and M. incurvatum can morphologically be differentiated from M. fulvum but not from each other. M. fulvum was the most abundant species isolated from the soils of Ahvaz; however, to comprehensively specify the distribution pattern of geophilic dermatophytes in the soils of this city further investigations are needed. Identification based on micro-morphometric is not effective for species distinction in M. gypseum complex, while molecular procedures based on sequencing of certain DNA regions are the most reliable and applicable strategies for this purpose.     

Antioxidants reveal an inverted U‐shaped dose‐response relationship between reactive oxygen species levels and the rate of aging in Caenorhabditis elegans

Reactive oxygen species (ROS) are potentially toxic, but they are also signaling molecules that modulate aging. Recent observations that ROS can promote longevity have to be reconciled with the numerous claims about the benefits of antioxidants on lifespan. Here, three antioxidants [N‐acetylcysteine (NAC), vitamin C, and resveratrol (RSV)] were tested on Caenorhabditis elegans mutants that alter drug uptake, mitochondrial function, and ROS metabolism. We observed that like pro‐oxidants, antioxidants can both lengthen and shorten lifespan, dependent on concentration, genotypes, and conditions. The effects of antioxidants thus reveal an inverted U‐shaped dose–response relationship between ROS levels and lifespan. In addition, we observed that RSV can act additively to both NAC and paraquat, to dramatically increase lifespan. This suggests that the effect of compounds that modulate ROS levels can be additive when their loci of action or mechanisms of action are sufficiently distinct.     

Oxamyl dipeptide caspase inhibitors developed for the treatment of stroke

Structural modifications were made to a previously described acyl dipeptide caspase inhibitor, leading to the oxamyl dipeptide series. Subsequent SAR studies directed toward the warhead, P2, and P4 regions of this novel peptidomimetic are described herein.