Identification and characterisation of novel CAR-T cells to target IL13Rα2 positive human glioma in vitro and in vivo.

BACKGROUND: Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB. METHODS: We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells. RESULTS: The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy. CONCLUSION: Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.

Prospects and Pitfalls of Plasma Complement C4 in Schizophrenia: Building a Better Biomarker.

Complex brain disorders like schizophrenia may have multifactorial origins related to mis-timed heritable and environmental factors interacting during neurodevelopment. Infections, inflammation, and autoimmune diseases are over-represented in schizophrenia leading to immune system-centered hypotheses. Complement component C4 is genetically and neurobiologically associated with schizophrenia, and its dual activity peripherally and in the brain makes it an exceptional target for biomarker development. Studies to evaluate the biomarker potential of plasma or serum C4 in schizophrenia do so to understand how peripheral C4 might reflect central nervous system-derived neuroinflammation, synapse pruning, and other mechanisms. This effort, however, has produced mostly conflicting results, with peripheral C4 sometimes elevated, reduced, or unchanged between comparison groups. We undertook a pilot biomarker development study to systematically identify sociodemographic, genetic, and immune-related variables (autoimmune, infection-related, gastrointestinal, inflammatory), which may be associated with plasma C4 levels in schizophrenia (SCH; n = 335) and/or in nonpsychiatric comparison subjects (NCs; n = 233). As with previously inconclusive studies, we detected no differences in plasma C4 levels between SCH and NCs. In contrast, levels of general inflammation, C-reactive protein (CRP), were significantly elevated in SCH compared to NCs (ANOVA, F = 20.74, p < 0.0001), suggestive that plasma C4 and CRP may reflect different sources or causes of inflammation. In multivariate regressions of C4 gene copy number variants, plasma C4 levels were correlated only for C4A (not C4B, C4L, C4S) and only in NCs (R Coeff = 0.39, CI = 0.01-0.77, R2 = 0.18, p < 0.01; not SCH). Other variables associated with plasma C4 levels only in NCs included sex, double-stranded DNA IgG, tissue-transglutaminase (TTG) IgG, and cytomegalovirus IgG. Toxoplasma gondii IgG was the only variable significantly correlated with plasma C4 in SCH but not in NCs. Many variables were associated with plasma C4 in both groups (body mass index, race, CRP, N-methyl-D-aspartate receptor (NMDAR) NR2 subunit IgG, TTG IgA, lipopolysaccharide-binding protein (LBP), and soluble CD14 (sCD14). While the direction of most C4 associations was positive, autoimmune markers tended to be inverse, and associated with reduced plasma C4 levels. When NMDAR-NR2 autoantibody-positive individuals were removed, plasma C4 was elevated in SCH versus NCs (ANOVA, F = 5.16, p < 0.02). Our study was exploratory and confirmation of the many variables associated with peripheral C4 requires replication. Our preliminary results point toward autoimmune factors and exposure to the pathogen, T. gondii, as possibly significant contributors to variability of total C4 protein levels in plasma of individuals with schizophrenia.

Complement C4 associations with altered microbial biomarkers exemplify gene-by-environment interactions in schizophrenia.

Schizophrenia is a complex brain disorder with genetic and environmental factors contributing to its etiology. Complement C4 genes are schizophrenia susceptibility loci and are activated in response to infections and gut microbiome imbalances. We hypothesize that C4 genetic susceptibility predisposes individuals to neuropathological effects from pathogen exposures or a microbiome in dysbiosis. In 214 individuals with schizophrenia and 123 non-psychiatric controls, we examined C4 gene copy number and haplotype groups for associations with schizophrenia and microbial plasma biomarkers. C4A copy number and haplotypes containing HERV-K insertions (C4A-long; C4AL-C4AL) conferred elevated odds ratios for schizophrenia diagnoses (OR 1.58-2.56, p < 0.0001), while C4B-short (C4BS) haplogroups conferred decreased odds (OR 0.43, p < 0.0001). Haplogroup-microbe combinations showed extensive associations with schizophrenia including C4AL with Candida albicans IgG (OR 2.16, p < 0.0005), C4AL-C4BL with cytomegalovirus (CMV) IgG (OR 1.79, p < 0.008), C4BS with lipopolysaccharide-binding protein (LBP) (OR 1.18, p < 0.0001), and C4AL-C4AL with Toxoplasma gondii IgG (OR = 17.67, p < 0.0001). In controls, only one haplogroup-microbe combination was significant: C4BS with CMV IgG (OR 0.52, p < 0.02). In schizophrenia only, LBP and CMV IgG levels were inversely correlated with C4A and C4S copy numbers, respectively (R2 = 0.13-0.16, p < 0.0001). C4 haplogroups were associated with altered scores of cognitive functioning in both cases and controls and with psychiatric symptom scores in schizophrenia. Our findings link complement C4 genes with a susceptibility to infections and a dysbiotic microbiome in schizophrenia. These results support immune system mechanisms by which gene-environmental interactions may be operative in schizophrenia.

Comparative Effectiveness of Plantar-Massage Techniques on Postural Control in Those With Chronic Ankle Instability.

CONTEXT: One of the major concerns after an acute lateral ankle sprain is the potential for development of chronic ankle instability (CAI). The existing research has determined that clinician-delivered plantar massage improves postural control in those with CAI. However, the effectiveness of self-administered treatments and the underlying cause of any improvements remain unclear. OBJECTIVES: To determine (1) the effectiveness of a self-administered plantar-massage treatment in those with CAI and (2) whether the postural-control improvements were due to the stimulation of the plantar cutaneous receptors. DESIGN: Crossover study. SETTING: University setting. PATIENTS OR OTHER PARTICIPANTS: A total of 20 physically active individuals (6 men and 14 women) with self-reported CAI. INTERVENTION(S): All participants completed 3 test sessions involving 3 treatments: a clinician-delivered manual plantar massage, a patient-delivered self-massage with a ball, and a clinician-delivered sensory brush massage. MAIN OUTCOME MEASURE(S): Postural control was assessed using single-legged balance with eyes open and the Star Excursion Balance Test. RESULTS: Static postural control improved (P ≤ .014) after each of the interventions. However, no changes in dynamic postural control after any of the interventions were observed (P > .05). No differences were observed between a clinician-delivered manual plantar massage and either a patient-delivered self-massage with a ball or a clinician-delivered sensory brush massage in any postural-control outcome. CONCLUSIONS: In those with CAI, single 5-minute sessions of traditional plantar massage, self-administered massage, and sensory brush massage each resulted in comparable static postural-control improvements. The results also provide empirical evidence suggesting that the mechanism for the postural-control improvements is the stimulation of the plantar cutaneous receptors.

Comparative Effectiveness of Plantar-Massage Techniques on Postural Control in Those With Chronic Ankle Instability.

CONTEXT: One of the major concerns after an acute lateral ankle sprain is the potential for development of chronic ankle instability (CAI). The existing research has determined that clinician-delivered plantar massage improves postural control in those with CAI. However, the effectiveness of self-administered treatments and the underlying cause of any improvements remain unclear. OBJECTIVES: To determine (1) the effectiveness of a self-administered plantar-massage treatment in those with CAI as well and (2) whether the postural-control improvements were due to the stimulation of the plantar cutaneous receptors. DESIGN: Crossover study. SETTING: University setting. PATIENTS OR OTHER PARTICIPANTS: A total of 20 physically active individuals (6 men and 14 women) with self-reported CAI. INTERVENTION(S): All participants completed 3 test sessions involving 3 treatments: a clinician-delivered manual plantar massage, a patient-delivered self-massage with a ball, and a clinician-delivered sensory brush massage. MAIN OUTCOME MEASURE(S): Postural control was assessed using single-legged balance with eyes open and the Star Excursion Balance Test. RESULTS: Static postural control improved (P ≤ .014) after each of the interventions. However, no changes in dynamic postural control after any of the interventions were observed (P > .05). No differences were observed between a clinician-delivered manual plantar massage and either a patient-delivered self-massage with a ball or a clinician-delivered sensory brush massage in any postural-control outcome. CONCLUSIONS: In those with CAI, single 5-minute sessions of traditional plantar massage, self-administered massage, and sensory brush massage each resulted in comparable static postural-control improvements. The results also provide empirical evidence suggesting that the mechanism for the postural-control improvements is the stimulation of the plantar cutaneous receptors.