Quantifying the limits of CAR T-cell delivery in mice and men

CAR (Chimeric Antigen Receptor) T cells have demonstrated clinical success for the treatment of multiple lymphomas and leukaemias, but not for various solid tumours, despite promising data from murine models. Lower effective CAR T-cell delivery rates to human solid tumours compared to haematological malignancies in humans and solid tumours in mice might partially explain these divergent outcomes. We used anatomical and physiological data for human and rodent circulatory systems to calculate the typical perfusion of healthy and tumour tissues, and estimated the upper limits of immune cell delivery rates across different organs, tumour types and species. Estimated maximum delivery rates were up to 10 000-fold greater in mice than humans yet reported CAR T-cell doses are typically only 10–100-fold lower in mice, suggesting that the effective delivery rates of CAR T cells into tumours in clinical trials are far lower than in corresponding mouse models. Estimated delivery rates were found to be consistent with published positron emission tomography data. Results suggest that higher effective human doses may be needed to drive efficacy comparable to mouse solid tumour models, and that lower doses should be tested in mice. We posit that quantitation of species and organ-specific delivery and homing of engineered T cells will be key to unlocking their potential for solid tumours.     

Real-time organic aerosol chemical speciation in the indoor environment using extractive electrospray ionization mass spectrometry

Understanding the sources and composition of organic aerosol (OA) in indoor environments requires rapid measurements, since many emissions and processes have short timescales. However, real-time molecular-level OA measurements have not been reported indoors. Here, we present quantitative measurements, at a time resolution of five seconds, of molecular ions corresponding to diverse aerosol-phase species, by applying extractive electrospray ionization mass spectrometry (EESI-MS) to indoor air analysis for the first time, as part of the highly instrumented HOMEChem field study. We demonstrate how the complex spectra of EESI-MS are screened in order to extract chemical information and investigate the possibility of interference from gas-phase semivolatile species. During experiments that simulated the Thanksgiving US holiday meal preparation, EESI-MS quantified multiple species, including fatty acids, carbohydrates, siloxanes, and phthalates. Intercomparisons with Aerosol Mass Spectrometer (AMS) and Scanning Mobility Particle Sizer suggest that EESI-MS quantified a large fraction of OA. Comparisons with FIGAERO-CIMS shows similar signal levels and good correlation, with a range of 100 for the relative sensitivities. Comparisons with SV-TAG for phthalates and with SV-TAG and AMS for total siloxanes also show strong correlation. EESI-MS observations can be used with gas-phase measurements to identify co-emitted gas- and aerosol-phase species, and this is demonstrated using complementary gas-phase PTR-MS observations.     

Lateral ion implant straggle and mask proximity effect

Lateral scattering of retrograde well implants is shown to have an effect on the threshold voltage of nearby devices. The threshold voltage of both NMOSFETs and PMOSFETs increases in magnitude for conventional retrograde wells, but for triple-well isolated NMOSFETs the threshold voltage decreases for narrow devices near the edge of the well. Electrical data, SIMS, and SUPREM4 simulations are shown that elucidate the phenomenon.     

Crystallization and preliminary X-ray diffraction studies of the soluble 14 kDa beta-galactoside-binding lectin from bovine heart

The soluble 14 kDa beta-galactoside-binding lectin from bovine heart, a member of the S-type lectin family, has been crystallized in a form suitable for X-ray diffraction analysis. The crystals, in the absence of a saccharide ligand, diffract beyond 2.5 A resolution. They are obtained from polyethylene glycol 6000 at pH 6.0. Crystals grow as monoclinic plates, space group P2(1), with cell dimensions: a = 35.47 A, b = 64.33 A, c = 58.78 A and beta = 91.7 degrees. The asymmetric unit contains two molecules related by a 2-fold non-crystallographic axis. Two lectin monomers in the asymmetric unit give a Vm of 2.4 A3/Da, i.e. a solvent content of approximately 50%. The complex of lectin with the saccharide ligand, N-acetyllactosamine, crystallizes in the space group P2(1)2(1)2 with cell dimensions: a = 63.55 A, b = 82.13 A and c = 62.39 A. Crystals of this complex diffract beyond 2.0 A resolution. Two complexes in the asymmetric unit lead to a Vm value of 2.8 A3/Da (57% solvent).     

An EIA method on single donor solubilized HLA antigens for the identification of anti-HLA antibodies

Primary biliary cirrhosis (PBC) is an immunologically mediated disease in which activated T lymphocytes attack and destroy epithelial cells in the small intralobular bile ducts of genetically susceptible patients. This article reviews the results of treatment of PBC with immunomodulatory agents. Results with drugs such as glucocorticoids, azathioprine, and chlorambucil have been disappointing because of either limited efficacy (azathioprine), toxicity (chlorambucil), or both (glucocorticoids). Colchicine improved tests of liver function in three prospective studies and was associated with improved survival for up to 4 years. However, survival benefits were lost at 8 years. Colchicine appears to slow the rate of progression of PBC but not to stop it. Preliminary results suggest that colchicine may have synergistic effects if used together with ursodeoxycholic acid, particularly in patients who are only partially responsive to ursodeoxycholic acid. Results with cyclosporine have been disappointing because of limited efficacy and predictable toxicity. The modest improvement in tests of liver function and survival are counterbalanced by the development of hypertension in some and worsening renal function in most. There is little beneficial effect on symptoms or histology. Results with methotrexate are promising. There are improvements in symptoms and tests of liver function that are equal to those seen with ursodeoxycholic acid and significant improvement in liver histology. Some patients, particularly those with striking inflammation and granulomas in portal triads, appear to have achieved sustained remission while on methotrexate. The effects of methotrexate are additive to those of ursodeoxycholic acid in patients whose blood tests have responded only partially to ursodeoxycholic acid. The most effective treatment of PBC will most likely use a combination of drugs such as ursodeoxycholic acid, colchicine, and methotrexate.     

Effect of preoperative supplementation with alpha-tocopherol and ascorbic acid on myocardial injury in patients undergoing cardiac operations

Augmentation of antioxidant defenses may help protect tissues against ischemia-reperfusion injury associated with operations involving cardiopulmonary bypass. In this study we examined the effect of pretreating patients with alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C) or placebo on injury to the myocardium. Seventy-six subjects undergoing elective coronary artery bypass grafting participated in a prospective, double-blind, placebo-controlled randomized trial, receiving either placebo or both 750 IU dl-alpha-tocopherol per day for 7 to 10 days and 1 gm ascorbic acid 12 hours before the operation. Plasma alpha-tocopherol concentrations, raised fourfold by supplementation, fell by 70% after the operation in the supplemented group and to negligible levels in the placebo group. There were no significant differences between the groups with respect to release of creatine kinase MB isoenzyme over 72 hours, nor in the reduction of the myocardial perfusion defect determined by thallium 201 uptake. Electrocardiography provided no evidence of a benefit from antioxidant supplementation. Thus the supplementation regimen prevented the depletion of the primary lipid soluble antioxidant in plasma, but provided no measurable reduction in myocardial injury after the operation.     

8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo

BACKGROUND: Myocardial reperfusion is believed to be associated with free radical injury. However, indexes of oxidative stress in vivo have been limited by their poor specificity and sensitivity. Isoprostanes are stable products of arachidonic acid formed in a nonenzymatic, free radical-catalyzed manner. We have developed a sensitive and specific assay for one of these compounds, 8-epi prostaglandin (PG) F2 alpha. METHODS AND RESULTS: To address its utility as an index of oxidative stress during coronary reperfusion, we measured urinary levels by gas chromatography/mass spectrometry in a canine model of coronary thrombolysis, in patients with acute myocardial infarction treated with thrombolytic therapy, and in patients after elective coronary artery bypass surgery. Urinary 8-epi PGF2 alpha was unchanged after circumflex artery occlusion in a canine model of coronary thrombolysis (n = 13; 437.2 +/- 56.4 versus 432.7 +/- 55.2 pmol/mmol creatinine) but increased significantly (P < .05) immediately after reperfusion (553.8 +/- 64.7 pmol/mmol). Urinary levels were increased (P < .001) in patients (n = 12) with acute myocardial infarction given lytic therapy (265.8 +/- 40.8 pmol/mmol) compared with age-matched control subjects (n = 20; 91.5 +/- 11.8 pmol/mmol) and patients with stable coronary disease (n = 20; 95.7 +/- 6.3 pmol/mmol). Preoperative levels rose from 113.2 +/- 11.8 to 248.2 +/- 86.3 pmol/mmol at 30 minutes into revascularization to 332.2 +/- 82.6 pmol/mmol by 15 minutes after global myocardial reperfusion (P < .05) and dropped to 181.2 +/- 50.4 pmol/mmol at 30 minutes and 120.2 +/- 9.9 pmol/mmol at 24 hours after bypass surgery (n = 5). Corresponding changes in spin adduct formation, found with electron paramagnetic resonance, were noted in 2 patients. CONCLUSIONS: These data support the hypothesis that free radical generation occurs during myocardial reperfusion. Measurement of isoprostane production may serve as a noninvasive index of oxidative stress.     

The pharmacokinetics and protein-binding of fusidic acid in patients with severe renal failure requiring either haemodialysis or continuous ambulatory peritoneal dialysis

Antibiotic treatment options for Burkholderia cepacia infection are limited because of high intrinsic resistance. The problem is complicated by development of cross-resistance between antibiotics of different classes. We isolated antibiotic-resistant mutants by stepwise exposure to chloramphenicol (Chlor) and to trimethoprim/sulphamethoxazole (T/S) for four B. cepacia strains: ATCC13945, Per (clinical isolate), Cas and D4 (environmental isolates). Chlor(r) mutants did not produce chloramphenicol acetyl-transferase. Cross-resistance, defined as greater than four-fold increase in MIC by microtitre dilution method, was consistently seen in both types of mutants. For chloramphenicol-resistant (Chlor[r]) and trimethoprim/sulphamethoxazole-resistant (Tr/Sr) mutants of B. cepacia ATCC13945 and Cas, no MIC change was seen for piperacillin, ceftazidime, rifampicin, gentamicin, tobramycin, polymyxin B or azithromycin. B. cepacia-Per and -D4 mutants showed cross-resistance to ceftazidime and to piperacillin. Comparison of outer membrane protein (OMP) profiles of B. cepacia and their mutants by SDS-PAGE revealed Tr/Sr) mutants to be deficient in a major OMP (molecular weight 39-47 kDa). Tr/Sr mutants also expressed additional OMPs not found in wild type strains at 75-77 kDa for B. cepacia-ATCC13945 and -Cas, and 20-21 kDa in B. cepacia-D4 and -Per. No OMP changes occurred in Chlor(r) mutants. Lipopolysaccharide (LPS) profiles of each type of mutant showed new high and low molecular weight LPS bands. Cross-resistance seems to be mediated by alterations in porin and LPS for Tr/Sr mutants, but only by LPS in Chlor(r) mutants.     

Drug rationalization: now for the hard part

In 1995 a national drug policy was finalized in Nepal. The authors outline the measures that were recommended for its implementation and the activities that have so far been undertaken. Much remains to be done, most notably in assisting, coordinating and supervising these activities.